CN101275135B - Antisense oligonucleotide for tissue-specific regulation of POKEMON gene expression and application thereof - Google Patents

Abstract

The present invention provides an antisense oligonucleotide of tissue-specific regulation POKEMON gene expression and its application. The antisense oligonucleotide is one of the following formulas: CxCxAxCxGyCyCyGyCyCyGyGyCyCyAxTxCxTx; CxGxCxTxGxAyCyGyAyAyGyTyCyGyAxTxCxTx; GxGxCxCxCyGyGyCyCyCyAyTyAyGyAxAxGxTx; TxTxCxAxGxGyTyCyGyTyAyGyTyTyGxTxGxGx; TxTxCxAxGyGyTyCyGyTyAyGyTyTyGxTxGxGx. X is bond between thiophosphate nucleoside. The antisense oligonucleotide of the invention is used for preparing gene therapeutic drug for curing disease caused by POKEMON high expression.

Description

The antisense oligonucleotide of specifically adjusting POKEMON gene expression and application thereof

Technical field

The present invention relates to a kind of antisense oligonucleotide and application thereof of specifically adjusting POKEMON gene expression.

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.

Natural oligonucleotide can not stable existence in serum, will be degraded fully in several minutes, and therefore, natural oligonucleotide can not be as the medicine of antisense therapy.

Summary of the invention

The purpose of this invention is to provide a kind of antisense oligonucleotide and application thereof of specifically adjusting POKEMON gene expression.

Antisense oligonucleotide provided by the present invention is, one of its molecular formula is following formula I to the formula IV:

5 ' CxCxAxCxGyCyCyGyCyCyGyGyCyCyAxTxCxTx 3 ' (formula I);

5 ' CxGxCxTxGyAyCyGyAyAyGyTyCyGyAxTxCxTx 3 ' (formula II);

5 ' GxGxCxCxCyGyGyCyCyCyAyTyAyGyAxAxGxTx 3 ' (formula III);

5 ' TxTxCxAxGyGyTyCyGyTyAyGyTyTyGxTxGxGx 3 ' (formula IV);

Wherein, formula I is to formula IV, and x all refers to key between the thiophosphatephosphorothioate nucleosides (phosphorothioate bond); Y all refers to key between the phosphodiester nucleosides (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 application of above-mentioned antisense oligonucleotide provided by the present invention is it at the preparation medicine that genetic expression is regulated, adjusted or suppresses to POKEMON, particularly treats the application in the medicine of the caused disease of POKEMON gene overexpression (as malignant tumour).

Described medicine is an effective constituent with above-mentioned antisense oligonucleotide.

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 50nmol/L---100nmol/L.

But antisense oligonucleotide specificity of the present invention suppresses the POKEMON expression of gene; Antisense oligonucleotide of the present invention has carried out the modification of phosphorothioate bond, and it all improves greatly in that stability of serum and cell, and toxic side effect is very little.Antisense oligonucleotide of the present invention can be used as effective constituent be used to prepare disease that treatment POKEMON high expression level causes gene therapy medicament.

Embodiment

The acquisition of the antisense oligonucleotide of embodiment 1, specifically adjusting POKEMON gene expression

1, the design of the prediction of POKEMON gene secondary structure and 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.At the reading frame of POKEMON gene, four sulfo-antisense oligonucleotide AS1～AS4 (following formula I-formula IV) that contain 18-mer have been designed in conjunction with its secondary structure.And in contrast with 18 nucleotide sequence AS5 not having a homology with any gene of people.

AS1:5 ' CxCxAxCxGyCyCyGyCyCyGyGyCyCyAxTxCxTx 3 ' (formula I);

AS2:5 ' CxGxCxTxGyAyCyGyAyAyGyTyCyGyAxTxCxTx 3 ' (formula II);

AS3:5 ' GxGxCxCxCyGyGyCyCyCyAyTyAyGyAxAxGxTx 3 ' (formula III);

AS4:5 ' TxTxCxAxGyGyTyCyGyTyAyGyTyTyGxTxGxGx 3 ' (formula IV);

AS5:5 ' AxTxGxTxAyGyTyCyGyTyCyTyGyCyCxTxAxGx 3 ' (formula V)

Wherein, formula I is to formula IV, 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.

AS1～AS4 can form the DNA/RNA heteroduplex with the mRNA of POKEMON gene, and it can be discerned by RNase H enzyme, degraded, thereby suppresses the POKEMON expression of gene.

2, solid phase synthesis POKEMON gene sulfo-antisense oligonucleotide

According to above-mentioned antisense oligonucleotide AS1, AS2, AS3, AS4, the molecular formula of AS5, use the ABI391DNA automatic DNA synthesizer DNA, synthesized sulfo-antisense oligonucleotide AS1～AS5 according to common G 3139 synthetic method, with the above-mentioned sulfo-antisense oligonucleotide AS1～AS5 for preparing, 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 sulfo-antisense oligonucleotide AS1～AS5 can give birth to the tailor-made molecular formula according to above-mentioned AS1, AS2, AS3, AS4, AS5 of worker's biotechnology Services Co., Ltd to Shanghai, by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd with the dNTP of α bit strip phosphorothioate bond as raw material, with the synthetic sulfo-antisense oligonucleotide AS1～AS5 of the automatic dna synthesizer of standard.

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%.

Embodiment 2, antisense oligonucleotide are tested the inhibition effect of POKEMON-pEGFP fusion rotein

1, the structure of the fusion plasmid of POKEMON and pEGFP

From HeLa Cells (available from American type culture collection, ATCC) in the clone obtain the reading frame segment of POKEMON, with its subclone to pEGFP-N ₂In the reporter plasmid, make it in mammalian cell, can express the POKEMON-EGFP fusion rotein.

1) cell cultures

People's uterus carcinoma (Hela) cell (available from American type culture collection, ATCC number:CCL-2) is 10% foetal calf serum 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), 84.00mgL-arginine monohydrochloride (L-Arginine-HCl), 63.00mg L-Gelucystine dihydrochloride (L-Cystine 2HCl), 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), 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

With PCR product EcoRV and HindIII double digestion that step 3) obtains, the clone advances the HindIII and the Sma I restriction enzyme site of pEGFP-N2 plasmid, obtains the pEGFP-N2-POKEMON fusion plasmid; 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 DNA25 μ l (0.4 μ g), 4.5ul10 * buffer K, 0.15ul HindIII (15U/ 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 can express the Pokemon-GFP fusion rotein in cell.

2, antisense oligonucleotide AS1, AS2, AS3, AS4 are to the inhibition effect of POKEMON-GFP fusion rotein

With liposome Lipofectamine 2000 (Invitrogen) as transfection reagent, with antisense oligonucleotide AS1 (formula I), AS2 (formula II), AS3 (formula III), AS4 (formula IV) and control oligonucleotide AS5 (ATGTAGTCGTCTGCCTAG) (concentration is 40nmol/L) and fusion plasmid pEGFP--N ₂-POKEMON cotransfection monkey kidney become fiber COS-7 cell (available from American type culture collection, ATCCnumber:CRL-1651), the 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,, include 10% foetal calf serum, the DMEM substratum of penicillin 100mg/ml and Streptomycin sulphate 100mg/ml, 5%CO in 37 ℃ 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 AS1 with embodiment 1 preparation, AS2, AS3, AS4 and AS5 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 AS1 that concentration is 40nmol/L in, AS2, AS3, AS4 and AS5 solution, get 2.5 μ l liposome Lipofectamine-2000 adding another one simultaneously and contain unparalleled anti-optimization substratum (the Opti-MEM I Reduced-SerumMedium 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 AS1, AS2, AS3, AS4 or AS5 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 AS5 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.

Above-mentioned steps 1) processing and step 2) processing experimental result as shown in Figure 1, wherein, among Fig. 1 A for the contrast (AS5) and pEGFP-N ₂-POKEMON plasmid co-transfection result, B are AS1 and pEGFP-N ₂-POKEMON plasmid co-transfection result, C are AS2 and pEGFP-N ₂-POKEMON plasmid co-transfection result, D are AS3 and pEGFP-N ₂-POKEMON plasmid co-transfection result, E are AS4 and pEGFP-N ₂-POKEMON plasmid co-transfection result.The result shows, in the COS-7 cell, and cotransfection pEGFP-N ₂-POKEMON and AS1, AS2, AS3 or AS4 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 AS2 (C among Fig. 1) and AS4 (E among Fig. 1) fluorescence intensity are weaker than the fluorescence intensity of AS1 (B among Fig. 1) and AS3 (D among Fig. 1), this explanation AS2 and AS4 suppress the inhibition efficient height of efficiency ratio AS1 and AS3.Use 5.02 pairs of fluorescence intensities of Image-pro plus v to carry out analytical calculation, AS2 and AS4 are to pEGFP-N ₂The inhibition efficient of the POKEMON-GFP fusion rotein that-POKEMON expresses is respectively 74% and 78%, same explanation, AS2 and AS4 are to the inhibition efficiency ratio AS1 of POKEMON-GFP fusion rotein and the inhibition efficient height of AS3, and it is most effective that AS4 suppresses, the result shows that AS4 can be used as the medicine of the disease that treatment Pokemon express to cause, as cancer etc.

Claims (8)

1. the antisense oligonucleotide of a specifically adjusting POKEMON gene expression, its molecular formula is suc as formula shown in the I:

5 ' CxCxAxCxGyCyCyGyCyCyGyGyCyCyAxTxCxTx 3 ' (formula I);

Wherein, among the formula I, 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.

The described antisense oligonucleotide of claim 1 preparation to POKEMON genetic expression regulate, adjust or suppress application in the medicine.

The described antisense oligonucleotide of claim 1 in the medicine of the caused disease of preparation treatment POKEMON gene overexpression application.

4. according to claim 2 or 3 described application, it is characterized in that: described medicine is to be effective constituent with the described antisense oligonucleotide of claim 1.

5. application according to claim 4 is characterized in that: described medicine is treated by subcutaneous administration.

6. application according to claim 5 is characterized in that: described medicine passes through topical treatment.

7. application according to claim 6 is characterized in that: described drug treatment combines with radiotherapy.

8. application according to claim 6 is characterized in that: described drug treatment combines with chemotherapy.

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