CN101275134B - Medicament for curing breast carcinoma and special antisense oligonucleotide therefor - Google Patents
Medicament for curing breast carcinoma and special antisense oligonucleotide therefor Download PDFInfo
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- CN101275134B CN101275134B CN2008100890563A CN200810089056A CN101275134B CN 101275134 B CN101275134 B CN 101275134B CN 2008100890563 A CN2008100890563 A CN 2008100890563A CN 200810089056 A CN200810089056 A CN 200810089056A CN 101275134 B CN101275134 B CN 101275134B
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Abstract
The present invention provides a medicine for curing mastopathy and its special antisense oligonucleotide. The molecular formula of the antisense oligonucleotide is: 5'TxTxCxAxGyGyTyCyGyTyAyGyTyTyGxTxGxGx 3', wherein, x is bonds between thiophosphate nucleosides; y is bonds between phosphodiester nucleosides; A is 2'-deoxyadenosine; T means thymidine; C means 2'-deoxycytidine; G means 2'-deoxyguanosine. The medicine for treating mastopathy is a medicine containing antisense oligonucleotide as effective component. The medicine using antisense oligonucleotide as effective component effectivelytreats human breast cancer.
Description
Technical field
The present invention relates to a kind of medicine and special antisense oligonucleotide thereof for the treatment of mammary cancer.
Background technology
POKEMON is a kind of newfound oncogene, and it can cause other oncogene outbreak, finally impels tumour to form.POKEMON can make cancer cells obtain anti-aging and dead ability, therefore is called as " master switch " of cancer.The more very important normal growth that is the disappearance of Pokemon in the cell can pair cell impacts, and this just provides possible for the specificity cancer therapy drug that designs low side effect.
Izant and Weintraub proposed antisense technology first in 1984, so-called antisense technology be exactly according between nucleic acid in conjunction with the base complementarity principle, with the antisense nucleic acid of synthetic or biosynthetic particular complementary or their chemical modification object and intracellular nucleic acid interaction with inhibition or seal its expression.The inactivation of some oncogene active or some cancer suppressor gene or lack relevant in the generation of malignant tumour and the cell.Antisense nucleic acid can be blocked the overexpression behind the oncogene activation specifically and not influence the normal function of other genes, utilize antisense technology to design and deleterious gene, mutator gene, improper expressing gene and mRNA complementary antisense nucleic acid thereof at present, to seal these genes or to suppress its expression.And the research of antisense nucleic acid treatment tumour is mainly used on tumor cell line, tumor animal and three levels of tumour patient.ISIS3521, ISIS5132, ISIS2503, G3139, GEM231 etc. have entered clinical experimental stage at present.
Summary of the invention
The purpose of this invention is to provide a kind of medicine for the treatment of mammary cancer and special use thereof instead with oligonucleotide.
Antisense oligonucleotide provided by the present invention is, suppresses the antisense oligonucleotide of POKEMON genetic expression, and its molecular formula is:
5’TxTxCxAxGyGyTyCyGyTyAyGyTyTyGxTxGxGx?3’
Wherein, x all refers to phosphorothioate bond; Y all refers to phosphodiester bond; A all refers to 2 '-Desoxyadenosine; T all refers to 2 '-thymidine; C all refers to 2 '-Deoxyribose cytidine; G all refers to 2 '-pancreatic desoxyribonuclease.
The medicine of treatment cancer provided by the present invention is the medicine that contains above-mentioned antisense oligonucleotide.
Described medicine is an effective constituent with above-mentioned antisense oligonucleotide.
Described medicine is the treatment cancer, particularly treats the medicine of mammary cancer.
Described medicine can be by passages through which vital energy circulates or subcutaneous administration treatment; Described medicine can pass through topical treatment.
Described drug treatment can combine with radiotherapy or with chemotherapy combined treatment disease.
The dosage scope of described drug treatment is 50-100nmol/L.
Can effectively the degrade mRNA of POKEMON gene in the MCF-7 cell of antisense oligonucleotide of the present invention reduces its expression at mRNA and protein level, and makes cell growth blocking-up at G
2/ M cycle, and then inducing apoptosis of tumour cell.Of the present invention is that the medicine of effective constituent is a kind of gene therapy medicament of effective treatment mammary cancer with the antisense oligonucleotide.
Description of drawings
Fig. 1 is antisense nucleotide ANP1, ANP2, ANP3, the ANP4 inhibition effect to the POKEMON-GFP fusion rotein
Fig. 2 be in the MCF-7 cell antisense nucleotide ANP4 to the inhibition effect of the mRNA of Pokemon
Fig. 3 be in the MCF-7 cell antisense nucleotide ANP4 to the inhibition effect of Pokemon and P53 protein level
Fig. 4 A is the influence of antisense nucleotide ANP0 to the growth of MCF-7 cell.
Fig. 4 B is the influence of antisense nucleotide ANP4 to the growth of MCF-7 cell.
Fig. 4 C is the influence of antisense nucleotide ANP0 to the growth of HepG-2 cell.
Fig. 4 D is the influence of antisense nucleotide ANP4 to the growth of HepG-2 cell.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
The acquisition of embodiment 1, POKEMON gene antisense oligonucleotide
1, the design of POKEMON gene antisense oligonucleotide
Utilize workstation (www.bioinfo.rpi.edu.cn/applications/mfold) that the secondary structure of the mRNA of POKEMON gene (NM 015898) is predicted.Reading frame at the POKEMON gene, designed Antisensedigonucleotsequence sequence ANP1, ANP2, ANP3, the ANP4 that contains 18 Nucleotide in conjunction with its secondary structure, and with 18 nucleotide sequence ANP0 not having a homology with any gene of people in contrast.
ANP0:5 ' AxTxGxTxAyGyTyCyGyTyCyTyGyCyCxTxAxGx 3 ' (formula I);
ANP1:5 ' CxCxAxCxGyCyCyGyCyCyGyGyCyCyAxTxCxTx 3 ' (formula II);
ANP2:5 ' CxGxCxTxGyAyCyGyAyAyGyTyCyGyAxTxCxTx 3 ' (formula III);
ANP3:5 ' GxGxCxCxCyGyGyCyCyCyAyTyAyGyAxAxGxTx 3 ' (formula IV);
ANP4:5 ' TxTxCxAxGyGyTyCyGyTyAyGyTyTyGxTxGxGx 3 ' (formula V);
Wherein, formula I is to formula V, and x all refers to phosphorothioate bond; Y all refers to 3,5 phosphodiester bonds; A all refers to 2 '-Desoxyadenosine; T all refers to 2 '-thymidine; C all refers to 2 '-Deoxyribose cytidine; G all refers to 2 '-pancreatic desoxyribonuclease.
Antisense oligonucleotide ANP1, ANP2, ANP3, ANP4 can form the DNA/RNA heteroduplex in the mRNA of POKEMON gene, and it can be by the identification of RNase H enzyme, degraded.
2, solid phase synthesis POKEMON gene antisense oligonucleotide
According to above-mentioned antisense oligonucleotide ANP0, ANP1, ANP2, ANP3, ANP4 molecular formula, and contrast ANP0 nucleotide sequence, with the dNTP of α bit strip phosphorothioate bond as raw material, with automatic dna synthesizer synthesising antisense scant nucleotide ANP1, ANP2, ANP3, ANP4 and the control oligonucleotide ANP0 of standard.With the above-mentioned antisense oligonucleotide ANP1 for preparing, ANP2, ANP3, ANP4 and control oligonucleotide ANP0 are respectively through the HPLC purifying, quantitative through ultraviolet spectrophotometer, concrete grammar is for accurately measuring the concentration of oligonucleotide with the DU800 ultraviolet spectrophotometer of Beckman Ku Erte (Beckmancoulter) company, in the cuvette of optical path 10mm, the optical extinction coefficient of single stranded oligonucleotide at the 260nm place is the 100 μ l microcuvettes of 20 μ g/ml/1OD. at optical path 10mm, add 98 μ l pure water, at fixed zero of 260nm and two wavelength places of 280nm, add 2 μ l oligonucleotide and mixings then, photoabsorption at 260nm and two wavelength places surveys of 280nm solution, the ratio of the A260/A280 of pure oligonucleotide is between 1.8 to 2.0, and be exactly the concentration (μ g/ μ l) of oligonucleotide in the OD value that 260nm surveyed this moment.Above-mentioned antisense oligonucleotide is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Detect the purity of oligonucleotide with denaturing polyacrylamide gel electrophoresis, the condition that experiment is adopted is that buffered soln is Tris (Tutofusin tris)--boric acid-EDTA, and the poly-propionic acid amide gel of the sex change of gel strength 30%, sequence by molecular sieve effect electrophoretic separation synthetic oligonucleotide and synthetic failure thereof, adopt the silver staining colour developing to present isolating collection of illustrative plates, this electrophoresis separating method can separate the oligonucleotide sequence that a nucleotide difference is only arranged, add sensitive silver staining developing technology, can detect the sequence of synthetic failure effectively. the result develops the color only has a colour developing band on the collection of illustrative plates, shows that the antisense oligonucleotide purity behind the purifying all is higher than 99%.
1, the structure of the fusion plasmid of POKEMON and pEGFP
The clone obtains the reading frame fragment of POKEMON from the HeLa Cells (available from American type culture collection, ATCC number:CCL-2), with its subclone to pEGFP-N
2In the reporter plasmid.Concrete grammar is as described below:
1) cell cultures
People's uterus carcinoma (Hela) cell (available from American type culture collection, be 10% foetal calf serum ATCC) with containing volume fraction, 100u/ml penicillin and 100u/ml Streptomycin sulphate and 0.2%NaHCO
3The DMEM nutrient solution, be 5% CO at 37 ℃, volume fraction
2The conventional cultivation under the condition.
DMEM (Dulbecco ' s Modified Eagle Medium) nutrient solution is very suitable for many mammalian cells and cultivates, be made up of component of inorganic salts, amino acid composition, vitamin component etc., its concrete prescription is for containing 200.00mg CaCl in every L substratum
2, 0.10mg Fe (NO
3)
39H
2O, 400.00mg KCl, 97.67mgMgSO
4, 6400.00mg NaCl, 3700.00mg NaHCO
3, 125.00mg NaH
2PO
4-H
2O, 4500mg glucose, phenol red 15.00mg (Phenol red), 110mg Sodium.alpha.-ketopropionate (Sodium Pyruvate), 84.00mg L-arginine monohydrochloride (L-Arginine-HCl), 63.00mg L-Gelucystine dihydrochloride (L-Cystine2HCl), 584.00mg L-glutaminate (L-Glutamine), 30.00mg glycine (Glycine), 42.00mg L-1-cystine mono hydrochloride (L-Histidine HCl-H2O), 105.00L-Isoleucine (L-Isoleucine), 105.00mg L-leucine (L-Leucine), 146.00mg lysine hydrochloride (L-Lysine-HCl), 30.00mg L-methionine(Met) (L-Methionine), 95.00mg L-Threonine (L-Threonine), 16.00mg L-tryptophane (L-Tryptophan), 104.00mg L-Tyrosine2Na 2H20,94.00mg L-Xie Ansuan (L-Valine), 4.00mg D-calcium pantothenate (D-Ca pantothenate), 4.00mg choline chloride 60 (Choline Chloride), 4.00mg folic acid (Folic Acid), 7.20mg inositol (i-Inositol), 4.00mg niacinamide (Niacinamide), and the 4.00mg vitamin B6 (vitamin B6 hydrochloride, PyridoxineHCl), 0.40mg Wei ShengsuB2 (riboflavin, 0.40), 4.00mg thiamine hydrochloride (Thiamine HCl), pH value 7.2-7.4.
2) extraction of cell total rna
Take out step 1) and cultivate people's uterus carcinoma (Hela) cell that obtains, nutrient solution (suspension cell needs centrifugal) is removed in suction, with an amount of PBS washing, add 1ml TRizol liquid, mixing, room temperature is placed 5min, liquid in the sucking-off culturing bottle is transferred to 1.5ml not to be had in the Rase enzyme EP pipe, and every pipe adds 0.2ml chloroform, concuss 15s, mixing, room temperature is placed 2-3min.12, the centrifugal 15min of 000g (2-8 ℃).Draw water in the EP of another 1.5ml pipe, add isopyknic Virahol, room temperature is placed 1min.12, the centrifugal 10min of 000g (2-8 ℃) has RNA precipitation to produce this moment, supernatant discarded, add 200-500ul 70% washing with alcohol after, sop up ethanol at the centrifugal 5min. of 7500g (4 ℃), blot liquid with little Tip.Precipitation seasoning 5-10 minute, DEPC treating water 20-30ul adds, and beats evenly with rifle, and 55-60 ℃ of water-bath allowed total RNA dissolve fully in 10 minutes, surveyed the OD value and obtained people's uterus carcinoma (Hela) cell total rna.
3) RT-PCR clone Pokemon
With step 2) people's uterus carcinoma (Hela) cell total rna of obtaining carries out reverse transcription, and reaction system 20ul comprises Oligo (dt) 1ul, people's uterus carcinoma (Hela) cell total rna 1ug, DEPC water places 70 ℃ of 5min, places 5min on ice then, add DEPC water, 5x damping fluid 4ul, 25mM MgCL
24ul adds 10mMdNTP1ul, RNA enzyme inhibitors 20U and ThermoScript II 1ul, and 25 ℃ of 5min, 42 ℃ of 60min in 70 ℃ of 15min termination reactions, obtain cDNA.Finish the back and carry out PCR as masterplate with cDNA.
Pokemon upstream region of gene primer: 5 '-CTTAAGCTTGCCACCATGGCCGGCGGCGTGG-3 ', downstream primer: 5 '-CATGATATCGGCGAGTCCGGCTGTGAAGTTAC-3 '.The PCR reaction system is Pokemon upstream and downstream each 10umol/L of primer, 5X buffered soln 10ul, 2mM dNTP 5ul, each 5ul of trimethyl-glycine and DMSO, 1ul cDNA, 2.5U/LPrimer star polysaccharase 1ul, total system 50ul.The PCR cycling condition: 98 ℃ of sex change 10s, 70 ℃ of renaturation 10s, 72 ℃ are extended 1min, 40 circulations.
Gained PCR product at 70v, is carried out agarose electrophoresis, and behind the 30min, glue places irradiation under the ultraviolet lamp, carefully dna fragmentation is downcut to be transferred in the 1.5ml EP pipe EP that weighs pipe, approximate definite volume.The density of supposing glue is 1g/ml, so the volume of gel can obtain by the following method: as gel thin slice quality is 0.2g, and then volume is 0.2ml, adds isopyknic NJ damping fluid then, mixture is placed 55-65 ℃ of water-bath 10min., dissolve fully to glue and mix.Transfer to glue after peptization separated and reclaim in the post, the centrifugal 1min of 8000g with SPW damping fluid repeated washing 2 times, adds the DNA elutriant at last the PCR product is washed, and 10, the centrifugal 1min of 000g.DNA output is calculated: 20 times of extracting diluted samples, survey the OD value under 260nm and 280nm.DNA concentration=A
260* 50 * extension rate.
4) structure of the fusion plasmid of POKEMON and pEGFP
The PCR product that step 3) is obtained is connected the fusion plasmid that obtains POKEMON and pEGFP with the pEGFP-N2 behind EcoRV and HindIII double digestion and HindIII and the Sma I double digestion; Concrete grammar is as follows:
The enzyme of the PCR product that 1. step 3) is obtained is cut: the enzyme of PCR product is cut to the 45ul system, reclaims DNA0.5ug, 4.5ul 10 * buffer K, 0.15ul HindIII (1.5U/ μ l), 0.15ul EcoRV (15U/ μ l), 15.2ulH
2O, 37 ℃ of enzymes were cut 3 hours.
2. pEGFP-N2 plasmid enzyme restriction and product purification reclaim: get 12 μ l pEGFP-N2 plasmids, 6 μ l10 * bufferM, 6 μ l HindIII, 5 μ l NaAc (3mM PH5.2), 150ul ethanol is in-20 ℃ of 10min, 10, the centrifugal 5min of 000g abandons supernatant, and precipitation adds 55 μ l TE dissolving.Add 55 μ l TE lysates after the end again, 8 μ l10 * buffer T, 8 μ l BSA, 6 μ l Sma I, 1 μ l Phosphoric acid esterase (CIAP), 37 ℃ of enzymes are cut 3 hours enzymes and are cut.After enzyme is cut end, add equal-volume NJ damping fluid, 8000g, 1min adds SPW liquid 8000g again, and 1min repeats once, adds the DNA elutriant at last, and 10,000g, 1min. is last that enzyme is cut purified product.
3. enzyme is cut being connected of purified pcr product and plasmid pEGFP-N2: linked system is 40ul, 2 μ l pEGFP-N2 (HindIII, Sma I), (HindIII EcoRV), places 56 ℃ of water-bath 4min to 34 μ lPCR purified products, 5min. adds 2 μ l T4 again and connects damping fluid on ice, 1 μ l 50mM ATP, 0.5 μ l T4 ligase enzyme, 16 ℃ of bindings are spent the night.
4. the conversion of the preparation of competent cell and connection product: get the JM109 bacterium liquid 800ul that spends the night, the centrifugal 5min of 3000g abandons supernatant, adds the fresh LB substratum of 50 μ l, with rifle piping and druming evenly, adds the CaCl of 2 μ l plasmid liquid (1 μ g/ μ l) and 2 μ l then
2, place 30min on ice, 42 ℃ of water-bath heat-shocked 60-120s, after the end, be positioned over 2-3min on ice, add 1mlLB liquid, and then shake 45min on the shaking table, after the end, at the centrifugal 5min of 3000g, remove the part supernatant liquor, remain about 100 μ l left and right sides liquid, add 12 μ l AMP, be coated with on the dropping flat board, the dull and stereotyped 30min that places of forward was inverted dull and stereotyped 7-8 hour then.
5. choose the clone and carry the positive colony cell for a short time: put on 1,2,3,4....20 20 LB flat board bottoms.Get 20 medium-sized centrifuge tubes, every pipe adds the LB substratum that 1.5ML contains (60 μ g/mL) kantlex, the different bacterium colonies line on the flat board that indicates numeral that grows after the above-mentioned connection product of picking transforms respectively with the toothpick of having sterilized (must be corresponding one by one), toothpick after the line is positioned in the medium-sized centrifuge tube (also must be on the pipe number peaceful model numeral corresponding one by one), culture dish after the line spent the night or places 10 hours for 37 ℃, spend the night on the medium-sized centrifuge tube shaking table (37 ℃) or the liquid of overnight incubation on the shaking table is transferred in the new little centrifuge tube, at the centrifugal 6min of 3000g, abandon supernatant liquid, precipitation is extracted plasmid, cut evaluation with BamH I enzyme, screening connects correct plasmid.BamH I enzyme cut identify that correct plasmid carries out sequencing result and shows that we have cloned and have obtained POKEMON reading frame sequence, and correctly put into pEGFP-N
2In the plasmid, order-checking is shown the correct segmental recombinant plasmid called after of the POKEMON pEGFP-N that contains
2-POKEMON.PEGFP-N
2-POKEMON plasmid enters in the cell through transfection, can express the Pokemon-GFP fusion rotein.
2, antisense oligonucleotide ANP1, ANP2, ANP3, ANP4 are to the inhibition effect of POKEMON-GFP fusion rotein
With liposome Lipofectamine 2000 (Invitrogen) as transfection reagent, with antisense oligonucleotide ANP1, ANP2, ANP3, ANP4 and control oligonucleotide ANP0 (concentration is 40nmol/L) and fusion plasmid pEGFP--N
2-POKEMON cotransfection monkey kidney becomes fiber COS-7 cell (available from American type culture collection ATCCnumber:CRL-1651), 24h fluorescence intensity.Concrete grammar is as described below:
1) pEGFP-N
2-POKEMON plasmid and antisense nucleotide cotransfection
Get the COS-7 cell of exponential phase of growth, in 37 ℃, include 10% foetal calf serum, the DMEM substratum of penicillin 100mg/ml and Streptomycin sulphate 100mg/ml is at 5%CO
2Incubator in cultivate.Cell transfecting the day before yesterday, cell suspends with trysinization and with the substratum of antibiotic-free, with every hole 1 * 10
5The density of individual COS-7 cell is spread into continuing cultivation 24 hours in 24 orifice plates.
Cultivate after 24 hours, antisense nucleotide ANP1 with embodiment 1 preparation, ANP2, ANP3, ANP4 and ANP0 are 40nmol/L with final concentration respectively and add unparalleled anti-optimization substratum (the Opti-MEM IReduced-Serum Medium of 50 μ l, GIBCO/BRL company product) the static 5min of mixing obtains the ANP1 that concentration is 40nmol/L in, ANP2, ANP3, ANP4 and ANP0 solution, get 2.5 μ l liposome Lipofectamine-2000 adding another one simultaneously and contain unparalleled anti-optimization substratum (the Opti-MEM IReduced-Serum Medium of 48.5 μ l, GIBCO/BRL company product) the static 5min of mixing in, after the end with the liposome after the dilution of above-mentioned preparation respectively with antisense thuja acid ANP1, ANP2, ANP3, ANP4 or ANP0 solution equal-volume mix, and add the pEGFP-N of 0.4 μ g respectively simultaneously
2-POKEMON plasmid will mix liquid then respectively and join above-mentioned shop according to the amount in 100 μ l/ holes and go in 24 orifice plates of COS-7 cell.With ANP0 and pEGFP-N
2-POKEMON plasmid co-transfection cell is done control group, detects the fluorescence intensity after above-mentioned experimental group and control group are cultivated 24h.
The experimental result of processing above-mentioned steps 1) as shown in Figure 1, the result shows, in the COS-7 cell, cotransfection pEGFP-N
2-POKEMON and ANP0, ANP1, ANP2, ANP3 or ANP4 antisense nucleotide.By fluorescence microscope, find that there is significant difference in four kinds antisense nucleotide to the inhibition of Pokemon.Can see that from Fig. 1 ANP2 (C among Fig. 1) and ANP4 (E among Fig. 1) fluorescence intensity are weaker than the fluorescence intensity of ANP1 (B among Fig. 1) and ANP3 (D among Fig. 1), this explanation ANP2 and ANP4 suppress the inhibition efficient height of efficiency ratio ANP1 and ANP3.Use 5.02 pairs of fluorescence intensities of Image-pro plus v to carry out analytical calculation, ANP2 and ANP4 are to pEGFP-N
2The inhibition efficient of the POKEMON-GFP fusion rotein that-POKEMON expresses is respectively 74% and 78%, same explanation, ANP2 and ANP4 are to the inhibition efficiency ratio ANP1 of POKEMON-GFP fusion rotein and the inhibition efficient height of ANP3, and it is most effective that ANP4 suppresses, and is confirmed as endogenous Study on Effect sequence with suppressing most effective ANP4.Wherein, A is contrast (ANP0) and pEGFP-N among Fig. 1
2-POKEMON plasmid co-transfection result, B are ANP1 and pEGFP-N
2-POKEMON plasmid co-transfection result, C are ANP2 and pEGFP-N
2-POKEMON plasmid co-transfection result, D are ANP3 and pEGFP-N
2-POKEMON plasmid co-transfection result, E are ANP4 and pEGFP-N
2-POKEMON plasmid co-transfection result.
With Lipofectamine 2000 (Invitrogen) as transfection reagent, antisense oligonucleotide ANP4 (concentration is 100nmol/L) transfection is arrived the MCF-7 cell (available from American type culture collection, ATCC number:HTB-22) in, concrete grammar is as described below:
Getting the MCF-7 of exponential phase of growth, is 10% foetal calf serum with including the quality percentage composition respectively, the DMEM substratum of penicillin 100mg/ml and Streptomycin sulphate 100mg/ml, and in 37 ℃, 5%CO
2Incubator in cultivate.Cell transfecting the day before yesterday, cell suspends with trysinization and with the DMEM substratum of antibiotic-free, with every hole 1 * 10
5Individual/cell density is spread into continuing cultivation 24 hours in 24 orifice plates.
Cultivate after 24 hours, with 5 μ l concentration is the antisense nucleotide ANP1 of 2 μ mol/L, ANP2, ANP3, ANP4 or ANP0 add the unparalleled anti-optimization substratum of 45 μ L (Opti-MEM I Reduced-Serum Medium respectively, GIBCO/BRL company product) the static 5min of mixing obtains antisense nucleotide ANP1 in, ANP2, ANP3, ANP4 or ANP0 solution, get 2.5 μ l Lipofectamine-2000 adding another one simultaneously and contain the unparalleled anti-optimization substratum of 48.5 μ l (Opti-MEM I Reduced-Serum Medium, GIBCO/BRL company product) the static 5min of mixing obtains liposome solutions in, finish the back liposome solutions respectively with antisense thuja acid ANP1, ANP2, ANP3, ANP4 or ANP0 solution equal-volume mix, to mix liquid then respectively joins above-mentioned shop according to the amount in 100 μ l/ holes and goes in 24 orifice plates of MCF-7 cell, continue to cultivate 24h or 48h, do control group with the cell of above-mentioned transfection ANP0 antisense nucleotide.
After the transfection 48 hours, with the POKEMON gene mRNA content in the RT-PCR detection transfectional cell, concrete grammar is as described below respectively:
(1) the MCF-7 cell after the taking-up transfection is inhaled and is removed nutrient solution, with an amount of PBS washing, adds 1ml TRizol liquid, mixing, and room temperature is placed 5min, and the interior liquid of sucking-off culturing bottle is transferred to 1.5ml not to be had in the Rase enzyme EP pipe.Every pipe adds the 0.2ml chloroform, concuss 15s, and mixing, room temperature is placed 2-3min.12, the centrifugal 15min of 000g (2-8 ℃).Draw water in the EP of another 1.5ml pipe, add isopyknic Virahol, room temperature is placed 1min.12, the centrifugal 10min of 000g (2-8 ℃) has the RNA precipitation to produce this moment.Supernatant discarded, add 200-500ul 70% washing with alcohol after, sop up ethanol at the centrifugal 5min. of 7500g (4 ℃), blot liquid with little Tip.Precipitation seasoning 5-10 minute, DEPC treating water 20-30ul adds, and beats evenly with liquid-transfering gun, and 55-60 ℃ of water-bath allowed total RNA dissolve fully in 10 minutes, surveyed OD value calculating total rna concentration.
(2) sxemiquantitative RT-PCR: reaction system 20 μ l comprise Oligo (dT) 1 μ l, the MCF-7 cell total rna 1 μ g after the transfection of said extracted, add DEPC water to 5 μ l, place 70 ℃ of 5min. to place 5min on ice, then, add DEPC water 4.5 μ l, 5x transcribes damping fluid 4 μ l, 25mM MgCl
24 μ l add 10mM dNTPl μ l, RNA enzyme inhibitors (40U/ μ l) 0.5 μ l and ThermoScript II 1 μ l, and 25 ℃ of 5min, 42 ℃ of 60min in 70 ℃ of 15min termination reactions, obtain cDNA.
With the above-mentioned cDNA that obtains as masterplate, with sxemiquantitative Pokemon upstream region of gene primer: 5 '-TGCAAGGTCCGCTTCACCAG-3 ', downstream primer: 5 '-GGCTGTGAAGTTACCGTCGGTG-3 ', the upstream primer of confidential reference items GAPDH: 5 '-CAACGTGTCAGTGGTGGACCTG-3 ', downstream primer: 5 '-TTACTCCTTGGAGGCCATGTGG-3 is that primer carries out pcr amplification.
The pcr amplification system is: last, the 10 μ mol/L downstream primers of Pokemon and GAPDH respectively are 2 μ l, 5XPrimerstar buffered soln 5ul, and 2mM dNTP 2 μ l,, 1 μ lcDNA, 2.5U/LPrimer star polysaccharase 0.5ul, water 7.5 μ l, cumulative volume 20ul.
The PCR cycling condition of sxemiquantitative Pokemon: 98 ℃ of sex change 10s, 60 ℃ of renaturation 10s, 72 ℃ are extended 40s, 24 circulations, the PCR cycling condition of GAPDH: 98 ℃ of sex change 10s, 60 ℃ of renaturation 10s, 72 ℃ are extended 30s, 22 circulations.The PCR product runs 1% agarose gel, dyes through pyridinium bromide (Ethidium Bromide).After taking pictures, picture can be used Quantity One software analysis.
The result as shown in Figure 2, the result shows that POKEMON gene mRNA content is 40% in the non-transfected cells in the cell of transfection of antisense oligonucleotides ANP4.The result shows that antisense oligonucleotide ANP4 has tangible Degradation to the POKEMON gene mRNA.Wherein, 0 among Fig. 2 among the A represents ANP0, and 1 represents ANP1, and 2 represent ANP2, and 3 represent ANP3, and 4 represent ANP4; Among Fig. 2 among the B+the normal MCF-7 cell of representative ,-represent ANP0.
According to the method for embodiment 3 with liposome Lipofectamine 2000 (Invitrogen) as transfection reagent, antisense oligonucleotide ANP4 (concentration is 100nmol/L) transfection is arrived the MCF-7 cell (available from American type culture collection, ATCC deposit number: HTB-22), transfection detected the POKEMO protein expression level with Westem-blot after 48 hours, and concrete grammar is as described below:
Get the MCF-7 cell of antisense nucleotide ANP4 transfection 48h in 6 orifice plates, with pancreatin room temperature digestion 3-5min, wash in 2000rpm with PBS, centrifugal 5min, abandon supernatant, look sedimentary amount and add suitable volumes (about 30-50 μ l) TNE (10mmol/L, 150mM NaCl, 0.5%NP-40,1mM) lysate places cracking 30min on ice.Lysate boils 10min 100 ℃ of water-baths, and last equal protein is run 10% polyacrylamide gel electrophoresis, 100v, 3h.After electrophoresis finished, by half-dried commentaries on classics film instrument, at 10v, 25min transferred to albumen on the film.After changeing the film end, add Ponceau S dyeing, the effect of determining to change film.With tap water flush away ponceau, will change film and be divided into two groups, every group of three gel pores add anti-Pokemon antibody (how anti-, as available from Sigma company, to dilute at 1: 4000) or antibodies against P 53 (monoclonal antibody available from Santa Cruz company, dilutes at 1: 500) respectively.Spend the night with an anti-incubated at room 1h or 4 ℃ respectively, one anti-hatch end after, with TBST washing 3 times, each 5min, then, the film of hatching Pokemon antibody is put into the TBST that adds the anti-rabbit of horseradish peroxidase labelled goat two anti-(dilutions in 1: 2000), the film of hatching P53 antibody is put into the TBST that adds goat anti-mouse two anti-(dilutions in 1: 2500), incubated at room 2h is respectively hatched after the end equally with TBST washing 3 times each 5min.Reagent with Super Signal WestPico Chemiluminesent substrate reagent box develops then, and uses x mating plate sensitization 5min in the darkroom.
The result as shown in Figure 3, the result shows that the POKEMON protein expression level shows that than having obvious reduction in the non-transfected cells antisense oligonucleotide ANP4 has the obvious suppression effect to the POKEMON expression of gene protein in the cell of transfection of antisense oligonucleotides ANP4.Have the obvious suppression effect because ANP4 expresses POKEMONP, removed POKEMONP to the restraining effect that P53 expresses, cause POKEMON downstream gene P53 to express and increase, P53 raises at protein level.Wherein, among Fig. 3 A be in the MCF-7 cell antisense nucleotide ANP4 to the inhibition effect of Pokemon protein level; Among Fig. 3 B in the MCF-7 cell antisense nucleotide ANP4 to the effect that increases of P53 protein level.Among Fig. 3 among A and the B "-" be ANP0; ANP4 all represents antisense nucleotide ANP4.
With Lipofectamine 2000 (Invitrogen) as transfection reagent, antisense oligonucleotide ANP4 (concentration is 100nmol/l) or ANP0 (concentration is 100nmol/l) are distinguished transfection in MCF-7 cell or liver cancer HepG-2 cell, after the transfection 48 hours, the method of singly dying with fluidic cell PI detects the variation of MCF-7 cell cycle, and concrete grammar is as described below:
Get the MCF-7 cell of antisense nucleotide ANP4 transfection 48h in 24 orifice plates or MCF-7 cell or the liver cancer HepG-2 cell (control group) of liver cancer HepG-2 cell (experimental group) and ANP0 transfection 48h, in PBS, wash 2 times, 500ul adds 70% ethanol and mixing then, spends down fixedly 2h in 4.The centrifugal 5min of sample 1000r/min abandons supernatant liquor during detection, the resuspended 5min of 400ul PBS.To remove agglomerating cell mass, under 1000r/min centrifugal 5 minutes again, abandon supernatant liquor with 300 eye mesh screen filtration cell suspensions.Add PI dye liquor 200ul/ pipe, lucifuge is 30 minutes under 4 degree.Transfer to liquid in the streaming pipe and add 400ul PBS.Utilize DNA distribution situation in the cells were tested by flow cytometry cell, single beam, excitation wavelength is 488nm.
The result at 100nmol/L ANP4 difference transfection MCF-7 and HepG-2 cell, can find (Fig. 4 B and Fig. 4 D), the G of MCF-7 and HepG-2 cell by the flow cytometer detection by figure as shown in Figure 4
2/ M phase DNA per-cent increases by 2.6% and 10% respectively, simultaneously G
0/ G
1The phase dna content also increases by 1.92% and 4.21% respectively.After our result shows drug effect Pokemon gene, cause cell generation cell-cycle arrest, particularly at G
2/ M the phase.And think extensively that at present cell is from G
2/ M the phase blocks in the process that withdraws from or afterwards, part cell generation apoptosis, part not apoptotic cells then directly enters " division stage death (mitotic death) ", and control group does not reach this effect, explanation is that the medicine of effective constituent can effectively be treated mammary cancer and liver cancer with ANP4, particularly mammary cancer is had extraordinary curative effect.Fig. 4 be with flow cytometer detect ANP4 to MCF-7 and HepG-2 (available from American type culture collection, ATCC number:HB 8065) cell growth influences figure, with the fluorescence of flow cytometer detection cell, transverse axis is represented fluorescence intensity, and the longitudinal axis is represented cell quantity.Wherein, Fig. 4 A and Fig. 4 B represent the AMCF-7 cell, and Fig. 4 C and Fig. 4 D group are represented the HepG-2 cell, Fig. 4 A and Fig. 4 C cell are handled with 100nmol/L oligonucleotide ANP0, organize in contrast, Fig. 4 B and Fig. 4 D cell are handled with 100nmol/L oligonucleotide ANP4, as experimental group.
Claims (7)
1. antisense oligonucleotide that suppresses POKEMON genetic expression, its molecular formula is:
5’TxTxCxAxGyGyTyCyGyTyAyGyTyTyGxTxGxGx?3’
Wherein, x all refers to key between the thiophosphatephosphorothioate nucleosides; Y all refers to key between the phosphodiester nucleosides; A all refers to 2 '-Desoxyadenosine; T all refers to thymidine; C all refers to 2 '-Deoxyribose cytidine; G all refers to 2 '-pancreatic desoxyribonuclease.
2. for being the medicine of the treatment cancer of effective constituent with the described antisense oligonucleotide of claim 1.
3. medicine according to claim 2 is characterized in that: described cancer is a mammary cancer.
4. medicine according to claim 3 is characterized in that: described medicine is by passages through which vital energy circulates or subcutaneous administration treatment.
5. medicine according to claim 4 is characterized in that: described medicine passes through topical treatment.
6. medicine according to claim 5 is characterized in that: described drug treatment combines with radiotherapy.
7. medicine according to claim 5 is characterized in that: described drug treatment combines with chemotherapy.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202900A (en) * | 1995-11-21 | 1998-12-23 | Icn药品公司 | Inhibition of tumor growth by antisense oligonucleotides for IL-8 and Il-8 receptor |
| CN1378552A (en) * | 1999-08-13 | 2002-11-06 | 乔治华盛顿大学 | Novel transcription factor BP1 |
| CN1704426A (en) * | 2004-05-26 | 2005-12-07 | 刘思源 | Cancer gene and its medical use |
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- 2008-04-15 CN CN2008100890563A patent/CN101275134B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202900A (en) * | 1995-11-21 | 1998-12-23 | Icn药品公司 | Inhibition of tumor growth by antisense oligonucleotides for IL-8 and Il-8 receptor |
| CN1378552A (en) * | 1999-08-13 | 2002-11-06 | 乔治华盛顿大学 | Novel transcription factor BP1 |
| CN1704426A (en) * | 2004-05-26 | 2005-12-07 | 刘思源 | Cancer gene and its medical use |
Non-Patent Citations (8)
| Title |
|---|
| Takahiro Maeda等.Role of the proto-oncogene Pokemon in cellulartransformation and ARF repression.Nature433 7023.2005,433(7023),278-285. |
| Takahiro Maeda等.Role of the proto-oncogene Pokemon in cellulartransformation and ARF repression.Nature433 7023.2005,433(7023),278-285. * |
| 李剑峰等.Pokemon 基因研究进展.实用肿瘤学杂志21 1.2007,21(1),98-100. |
| 李剑峰等.Pokemon 基因研究进展.实用肿瘤学杂志21 1.2007,21(1),98-100. * |
| 邓一静等.Pokemon 基因 siRNA 逆转录病毒重组表达质粒的构建及鉴定.免疫学杂志22 2.2006,22(2),203-209. |
| 邓一静等.Pokemon 基因 siRNA 逆转录病毒重组表达质粒的构建及鉴定.免疫学杂志22 2.2006,22(2),203-209. * |
| 邬赟斌等.原癌基因Pokemon的研究进展.中国肿瘤生物治疗杂志13 2.2006,13(2),153-155. |
| 邬赟斌等.原癌基因Pokemon的研究进展.中国肿瘤生物治疗杂志13 2.2006,13(2),153-155. * |
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