CN1476899A - Multidrug-resistant RNA interference drugs against tumors - Google Patents
Multidrug-resistant RNA interference drugs against tumors Download PDFInfo
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Abstract
The present invention discloses an antitumor multi-drug drug-resistant RNA interference drug, provides a group of nucleotide sequences of interference RNA of therapeutic drug pointed at drug-resistant gene mdr-1 and P-glycoprotein (P-gp) expression and function for resisting leucosis and other tumor. It has the target antitumor cell multidrug drug-resistant (mdr-1) gene and P-glycoprotein (P-gp) expression and function. It raises the sensitivity of leucosis cell conventional chemical therapeutic drug and enhances the action of chemical therapeutic drug for killing malignant tumor cell of hemopoietic system.
Description
Technical field
The present invention relates to anti-tumor drug, especially antineoplastic multidrug resistance RNA interference medicament.
Background technology
(multidrug resistance MDR) is the significant obstacle that malignant tumor conventional chemotherapies such as leukemia are difficult to obtain long-term remission to multidrug resistance.Studies show that (p-glycoprotein is to cause mainly mechanism of tumor cell multidrug resistance in the tumor cell surface overexpression P-gp), also is the good target spot of reversing drug resistance by the p-glycoprotein of mdr-1 gene code.The medicine of the energy reversion MDR of having reported at present has tens of kinds more than, but toxicity is bigger under the active drug concentration, produces serious adverse reaction, has limited its clinical practice.Therefore carry out the focus that target treatment is domestic and international multi-medicine drug resistance of leukaemia reverse research at mdr-1 gene and albumen thereof.
(RNA interference RNAi) is a kind of effective ways that seal gene expression that newly-developed gets up in the RNA interference.It adopts and long RNA interfering (the small interfering RNA of the homologous 21-23 nucleotide of genes of interest, siRNA) transfection is to target cell, induce silencing complex (RISC) with intracellular restriction endonuclease formation, RISC is that template is discerned its homologous genes mRNA specifically and it is carried out laddering shearing with siRNA, form strong effectively water fall effect, the specific mRNA degraded of induced sequence, the defective phenotype of cell performance specific gene.This strategy can be closed the cancer gene, only has a base mutation then to lose the RNA interference effect, and is very little to normal impact cell, high specificity.
Summary of the invention
Technical problem to be solved by this invention provides a kind of antineoplastic multidrug resistance RNA interference medicament, adopt the RNAi technology, siRNA at the mdr-1 gene reverses multiple medicine-resistant cell line cell drug-resistant phenotype in the mRNA level, can effectively suppress the expression of multidrug resistance gene mdr-1.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of antineoplastic multidrug resistance RNA interference medicament, carry out target treatment at multidrug resistance gene mdr-1 and expressing protein thereof, have targeting antitumor cell multidrug resistance mdr-1 gene and P-P-glycoprotein expression and function, interference RNA sequence is the siRNA sequence of 3 sections 21 bases choosing at mdr-1 gene mRNA different loci, be referred to as si-mdr1, si-mdr2 and si-mdr3, described RNA interference medicament is si-mdr1, si-mdr2, in three nucleotide sequences of si-mdr3 any one, two or three combination, si-mdr1, si-mdr2, three nucleotides sequences of si-mdr3 are classified as:
si-mdr1： CUGUACUGGUCCAUACGGAUU
UUGACAUGACCAGGUAUGCCU
si-mdr2： CGCCGAGGCUAUGUACCAAUU
UUGCGGCUCCGAUACAUGGUU
si-mdr3： CCUCCGGUUGUAUGUACGGUU
UUGGAGGCCAACAUACAUGCC
The target site of described si-mdr1, si-mdr2, si-mdr3 is: the target site of si-mdr1: 5 '-AAGACAUGACCAGGUAUGCCU-3 ', and--------(2472-2492) target site of si-mdr3: 5 '-AAGGAGGCCAACATACATGCC-3 '----(3564-3584) for (865-885) target site of si-mdr2: 5 '-AAGCGGCTCCGATACATGGTT-3 '
Route of administration can adopt: directly naked DNA injection, liposome dna direct injection, Jin Bao are carried a kind of in the plasmid DNA method by DNA particle gun blast technique, breeding unsoundness antibacterial.
The siRNA of the present invention's development can effectively suppress the expression of multidrug resistance gene MDR-1, provides experimental evidence and effective new drug for the RNAi technology is applied to leukemic treatment.
Description of drawings
Fig. 1 is the pcr amplification product electrophoretogram of K562/A02 cell mdr-1 behind the siRNA processing 24h of the present invention.
Fig. 2 is the result that siRNA of the present invention handles the influence that P-gp is expressed.
Among the figure, the 1:si-neg group; The 2:si-mdr1 group; The 3:si-mdr2 group; The 4:si-mdr3 group; The 5:PCR negative control.
The specific embodiment
Below in conjunction with the drawings and specific embodiments antineoplastic multidrug resistance RNA interference medicament of the present invention is described in further detail:
The present invention adopts the RNAi technology, and (smallinterfering RNA siRNA) reverses multiple medicine-resistant cell line K562/A02 cell drug-resistant phenotype at the siRNA of mdr-1 gene in the mRNA level.K562 cell line is the cell line from the erythroleukemia patient, and the cell line of the adriamycin-resistant (ADM) of K562/A02 cell line through repeatedly screening.
SiRNA sequence involved in the present invention is the siRNA sequence of 3 sections 21 bases choosing at mdr-1 gene mRNA different loci, is referred to as si-mdr1, si-mdr2 and si-mdr3.Other establishes random sequence as negative control (si-neg).The siRNA sequence is as follows: the target site of si-mdr1: 5 '-AAGACAUGACCAGGUAUGCCU-3 '----(865-885)
s-mdr1： CUGUACUGGUCCAUACGGAUU
The target site of UUGACAUGACCAGGUAUGCCUsi-mdr2: 5 '-AAGCGGCTCCGATACATGGTT-3 '----(2472-2492)
si-mdr2： CGCCGAGGCUAUGUACCAAUU
The target site of UUGCGGCUCCGAUACAUGGUUsi-mdr3: 5 '-AAGGAGGCCAACATACATGCC-3 '----(3564-3584)
si-mdr3： CCUCCGGUUGUAUGUACGGUU
UUGGAGGCCAACAUACAUGCCsi-neg： 5’-AATAGGATACGTGACGCTATG-3’
UCCUAUGCACUGCGAUACUU
UUAGGAUACGUGACGCUAUG
The used K562/A02 cell line of the present invention is the cell line of adriamycin-resistant (ADM) erythroleukemia.Cell inoculation is in RPMI1640 (the Gibco BRL company product) culture fluid that contains 10% calf serum, and placing 37 ℃, volume fraction is 5% CO
2The conventional cultivation in the incubator, no medicine cultivated for two weeks before the experiment.Optimize transfection conditions, respectively si-mdr1, si-mdr2, si-mdr3, si-neg are added the K562/A02 cell culture fluid with the final concentration of 200nmol, detect in hatching back 24～48h harvesting.
The present invention is from mRNA and two levels of protein level detect si-mdr1, si-mdr2 and si-mdr3 suppresses the multidrug resistance gene expression and improves the effect of drug-resistant leukemia cell to the sensitivity of chemotherapeutics.RT-PCR and the quantitative analysis of PCR product are adopted in the detection of mRNA.Technology path: 1) RT-PCR: extract total RNA by the total RNA extraction agent of TRIzol box.Electrophoresis is identified the quality of RNA, and the ultraviolet light spectrophotometer is quantitative.Get total RNA 2.5 μ g, add 50 μ l reverse transcription reaction systems, use SuperScript
TMIIReverse transcription test kit (available from Invitrogen company), by specification operation carrying out reverse transcription.The PCR reaction system is (reagent is available from Invitrogen company) routinely.Cycling condition: 94 ℃ of degeneration 45 seconds, 58 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, extended at last 10 minutes.
Amplified production with β-actin is the confidential reference items contrasts in addition.Amplimer sequence and amplified fragments are as follows: the mdr-1 forward primer: 5 ' TTACACGTGGTTGGAAGC-3 '
Downstream primer: 5 ' CATAGATCAGCAGGAAAG-3 ', amplified fragments 300bp.β-actin forward primer: 5-' GTGGGGCGCCCCAGGCACCA-3 '
Downstream primer: 5 '-CTCCTTAATGTCACGCACGATTC-3 ', amplified fragments 548bp.
The PCR product is quantitative: the amplified production of getting 10 μ l carries out electrophoresis on the agarose gel electrophoresis of 15g/L, with PCR mark (magnificent company) as the molecular mass standard, voltage 100V, 20 minutes.Ethidium bromide staining, video picture on the ultraviolet reflectance instrument, photograph.Carry out the sxemiquantitative (expression that reflects mdr-1 mRNA with the ratio of mdr-1/ β-actin cDNA) of genes of interest with scanning analysis.
Flow cytometry is adopted in the detection of multidrug resistance gene mdr-1 expression, detects the expression of P-gp.That collects 48h respectively organizes the K562/A02 cell, and PBS washing 2 times adds P-gp monoclonal antibody 10 μ l (Neomarkers company product), and 4 ℃, 30 minutes; The PBS washing adds two of FITC labelling and resists, and 4 ℃, 30 minutes.The PBS washing, last machine testing.
The present invention adopts MTT to detect the 50 3nhibitory dose IC of amycin to drug treating leukaemia and contrast K562 cell
50, be evaluation index with reversing efficient relatively, reverse efficient=(IC relatively
50A-IC
50B)/(IC
50A-IC
50C).IC
50A is the IC of K562/A02
50, IC
50B is the IC after the siRNA effect
50, IC
50C is the IC of K562
50The IC of resistance factor RF=K562/A02
50The IC of/K562
50
The present invention also detects the mensuration of daunorubicin accumulation in the cell with flow cytometer, the cell furnishing 1 * 10 that will handle through RNAi
6The daunorubicin of/ml and 1g/ml is hatched jointly in 37 ℃, takes out after 1 hour, and ice bath is to stop the effect of daunorubicin.Detect daunorubicin excited fluorescent intensity in the cell with flow cytometer, reflect DNR in intracellular concentration, excitation wavelength 488nm, emission wavelength 550nm.With undressed K562/A02 cell is blank.
Carry out the focus that target treatment is domestic and international multi-medicine drug resistance of leukaemia reverse research at mdr-1 gene and albumen thereof, concentration when effective reversing drug resistance such as inversion agent verapamil now commonly used, cyclosporin A produces serious adverse reaction to human body, has limited its clinical practice.Because the RNAi that the present invention uses has the sequence specificity of height, can make the specific gene silence specifically, obtain afunction or reduce sudden change, no matter be therefore, or the genetic treatment of tumor aspect all have broad application prospects at functional genome research.
The present invention has designed 3 at the different target sites of mdr-1 gene and has disturbed the siRNA sequences, in order to the MDR of reversing multi-medicine drug resistance of leukaemia cell strain according to the siRNA user guide design principle of Elbashir etc.K562/A02 is the acute transformation of chronic myelocytic leukemia cell line with typical multidrug resistance feature, high expressed mdr-1 gene.IC
50, drug level, p170 express and the mRNA relative quantity detects the sealing mdr-1 gene that confirms that 3 sequences can be in various degree in the cell, reverse the cell drug-resistant phenotype.These inventions confirm that siRNA is expected to become the drug-fast effective means of reversing tumor.The siRNA of invention can use separately or several siRNA associatings, can also unite with other inversion agent such as antisensenucleic acids and make pharmaceutical composition, is used for leukemia and other tumor treatment.
The route of administration of said gene sequence, spendable method has following several:
1, direct naked DNA injection
(1) new jet injecting systems discharges naked DNA treatment sequence, can adopt a kind of new jet injecting systems, and naked DNA is discharged in the intravital lung tumor.Wal plucked instrument (W.Walther) doctor and colleague have developed a kind of portable " high speed jet syringe " system, provide another selection outside viral vector and liposome gene delivery system.Researcher uses 3 sections naked DNAs of this system's injection in the Lewis lung tumor of mice, and first plasmid expression beta galactose kinase gene (LacZ) is expressed green fluorescence (GFP) gene for second, expresses human tumor necrosis factor-alpha (TNF-α) for the 3rd.Each mice is all accepted 5 injections, and pressure is 3Pa, discharges 3～5 microlitre plasmid DNA.Wal plucked instrument doctor etc. reports that at " gene therapy " (Gene Therapy) gene on the tumor of injection back is wide expression.LacZ and GFP gene expression became obviously in injection in back 48 hours, reached peak value at 72～96 hours; LacZ expresses and TNF-α secretion occurred at 24～120 hours.They think that " these discoveries have proved the suitability of jet injection, use the naked DNA of minimum dose to carry out gene therapy for cancer when promptly transmitting gene to tumor in vivo.”
(2) naked DNA direct injection: naked plasmid dna is injected directly into the muscle, Intradermal of body, subcutaneous, mucosa, intravenous.This method is simple.
2, liposome dna direct injection: lipid physical ability and the histiocyte generation film of parcel DNA merge, and DNA is taken in, and have reduced the destruction of nuclease to DNA.Injecting pathway is with the naked DNA direct injection.
3, Jin Bao is by DNA particle gun blast technique: plasmid DNA is coated on golden micropartical surface, makes bag be penetrated histiocyte at a high speed by the golden micropartical of DNA with particle gun.
4, the breeding unsoundness antibacterial is carried the plasmid DNA method: select a kind of antibacterial that enters certain histoorgan easily, its breeding gene is removed, use the plasmid DNA transform bacteria then, after these antibacterials enter certain histoorgan, because irreproducible, then self cracking and discharge plasmid DNA.That is: attenuation salmonella that can be oral.The situation that embodiment 1.RNAi expresses drug resistant gene mdr-1 mRNA
The K562/A02 cell detects after siRNA handles 24h, compare with negative control, si-mdr1 organizes mRNA down-regulated expression (17.23 ± 2.47) % (p＞0.05), si-mdr2 and si-mdr3 handle the mRNA that detects mdr-1 after 24 hours and express obviously minimizing, be respectively (38 ± 1.23) % and (58 ± 1.54) % (p＜0.05), see Fig. 1.
Embodiment 2.P-gp detection of expression
Flow cytometer detects and shows behind the siRNA effect 48h, three groups of expression that suppress p170 in varying degrees, the positive rate that si-mdr1 effect group p170 expresses is by (76.0 ± 1.03) % before handling, reduce to (56.72 ± 1.41) % after the processing, si-mdr2 effect group is reduced to (42.70 ± 1.17) % (p＜0.05); And the positive rate that si-mdr3 effect group p170 expresses significantly reduces, and is (19.57 ± 1.94) % (p＜0.01).The p170 The positive expression rate did not have obvious change before and after the si-neg experimental group was handled: (76.0 ± 1.03) % before handling, handle back (74.6 ± 0.75) % (p＞0.05), and see Fig. 2.
RNAi handles back K562/A02 cell drug sensitivity and changes: IC
50The phalangeal cell suppression ratio is the concentration of 50% o'clock chemotherapeutics, and after si-mdr2 and the si-mdr3 effect, K562/A02 increases the sensitivity of chemotherapeutics ADM as shown in Table 1, and prompting RNAi can recover the sensitivity of K562/A02 to chemotherapeutics.Embodiment 3.siRNA is to the drug-fast reverse effect of K562/A02 cell
Table 1 is the result comparison of siRNA to the drug-fast reverse effect of K562/A02 cell.Each experiment repeats 3 times, establishes 3 parallel holes at every turn, gets average.Si-mdr1, si-mdr2 and si-mdr3 all have the multi-medicine tolerant reversal effect, and the effect of si-mdr3 is the most remarkable.
Table 1
IC
50(μ g/ml) reverses efficient relatively
(％)
K562 0.05 /
K562/A02 4.47 /
si-neg 4.28 4.29
si-mdr1 3.67 18.10
si-mdr2 2.53* 43.89
Si-mdr3 1.36** 70.36 annotates: compare p value: *＜0.05, * *＜0.01 with the K562/A02 groups of cells.Embodiment 4.RNAi handles the variation of DNR accumulation in the cell of back
Handle K562/A02 and K562 cell with si-mdr, find that DNR all has the trend of increase than its fluorescence intensity before handling and positive rate in the K562/A02 cell, and significant difference is arranged.But K562 compares with parental cell, and fluorescence intensity and positive rate are still lower, see Table 2.
Table 2:si-RNA handles ADM cumulative change in the cell of back
Average fluorescent strength positive rate (%)
K562 86.35 97.18
Si-neg 37.66 27.76
si-mdr1 41.05 34.57
si-mdr2 57.88* 61.40
Si-mdr3 64.48** 78.07** annotates: compare p value: *＜0.05, * *＜0.01 with the K562/A02 groups of cells 1.; 2. each experiment repeats 3 times, gets average.
Claims (3)
1, a kind of antineoplastic multidrug resistance RNA interference medicament, carry out target treatment at multidrug resistance gene mdr-1 and expressing protein thereof, have targeting antitumor cell multidrug resistance mdr-1 gene and P-P-glycoprotein expression and function, it is characterized in that interference RNA sequence is the siRNA sequence of 3 sections 21 bases choosing at mdr-1 gene mRNA different loci, be referred to as si-mdr1, si-mdr2 and si-mdr3, described RNA interference medicament is si-mdr1, si-mdr2, in three nucleotide sequences of si-mdr3 any one, two or three combination, si-mdr1, si-mdr2, three nucleotides sequences of si-mdr3 are classified as:
si-mdr1： CUGUACUGGUCCAUACGGAUU
UUGACAUGACCAGGUAUGCCU
si-mdr2： CGCCGAGGCUAUGUACCAAUU
UUGCGGCUCCGAUACAUGGUU
si-mdr3： CCUCCGGUUGUAUGUACGGUU
UUGGAGGCCAACAUACAUGCC
2, antineoplastic multidrug resistance RNA interference medicament according to claim 1,--------(2472-2492) target site of si-mdr3: 5 '-AAGGAGGCCAACATACATGCC-3 '----(3564-3584) for (865-885) target site of si-mdr2: 5 '-AAGCGGCTCCGATACATGGTT-3 ' to it is characterized in that the target site of described si-mdr1, si-mdr2, si-mdr3 is: the target site of si-mdr1: 5 '-AAGACAUGACCAGGUAUGCCU-3 '
3, antineoplastic multidrug resistance RNA interference medicament according to claim 1 is characterized in that route of administration can adopt: directly naked DNA injection, liposome dna direct injection, Jin Bao are carried a kind of in the plasmid DNA method by DNA particle gun blast technique, breeding unsoundness antibacterial.
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| CN031303714A CN1217700C (en) | 2003-07-08 | 2003-07-08 | Multi-medicine medicine-resistant RNA interference medicine for resisting tumor |
| PCT/CN2004/000762 WO2005002594A1 (en) | 2003-07-08 | 2004-07-07 | An anti-tumor rna interference drug for multidrug resistance |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100344330C (en) * | 2004-09-22 | 2007-10-24 | 广州拓谱基因技术有限公司 | Targeted small interference RNA formulation for treating viral Hepatitis B and its preparation |
| CN100344331C (en) * | 2004-09-22 | 2007-10-24 | 广州拓谱基因技术有限公司 | Targeted small interference RNA formulation for preventing and treating Hepatitis C and its preparation method |
| CN1704123B (en) * | 2004-06-01 | 2010-06-02 | 广州拓谱基因技术有限公司 | Little interfered RNA preparation for internal preventing or curing respiratory system diseases and screening method thereof |
| CN101897982A (en) * | 2009-05-31 | 2010-12-01 | 苏州圣诺生物医药技术有限公司 | SiRNA medicinal composition for treating cancers |
| CN102526762A (en) * | 2012-01-19 | 2012-07-04 | 广州医学院 | Application of ALC1 to preparation of leukaemia drug resistance reversing agent and as drug-resistant leukaemia diagnosing reagent |
| CN104189920A (en) * | 2014-07-31 | 2014-12-10 | 清华大学 | Gene composition h-R3/PAMAM siRNA for reversing multidrug resistance of tumors and application of gene composition |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1789447B1 (en) | 2004-08-16 | 2012-04-25 | Immune Disease Institute, Inc. | Method of delivering rna interference and uses thereof |
| AU2011336467A1 (en) | 2010-12-01 | 2013-07-04 | Spinal Modulation, Inc. | Agent delivery systems for selective neuromodulation |
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| DE10230996A1 (en) * | 2001-10-26 | 2003-07-17 | Ribopharma Ag | Method for inhibiting viral replication, useful particularly for treating hepatitis C infection, by altering the 3'-untranslated region of the virus |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1704123B (en) * | 2004-06-01 | 2010-06-02 | 广州拓谱基因技术有限公司 | Little interfered RNA preparation for internal preventing or curing respiratory system diseases and screening method thereof |
| CN100344330C (en) * | 2004-09-22 | 2007-10-24 | 广州拓谱基因技术有限公司 | Targeted small interference RNA formulation for treating viral Hepatitis B and its preparation |
| CN100344331C (en) * | 2004-09-22 | 2007-10-24 | 广州拓谱基因技术有限公司 | Targeted small interference RNA formulation for preventing and treating Hepatitis C and its preparation method |
| CN101897982A (en) * | 2009-05-31 | 2010-12-01 | 苏州圣诺生物医药技术有限公司 | SiRNA medicinal composition for treating cancers |
| CN102526762A (en) * | 2012-01-19 | 2012-07-04 | 广州医学院 | Application of ALC1 to preparation of leukaemia drug resistance reversing agent and as drug-resistant leukaemia diagnosing reagent |
| CN104189920A (en) * | 2014-07-31 | 2014-12-10 | 清华大学 | Gene composition h-R3/PAMAM siRNA for reversing multidrug resistance of tumors and application of gene composition |
| CN104189920B (en) * | 2014-07-31 | 2017-02-01 | 清华大学 | Gene composition h-R3/PAMAM siRNA for reversing multidrug resistance of tumors and application of gene composition |
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| CN1217700C (en) | 2005-09-07 |
| WO2005002594A1 (en) | 2005-01-13 |
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