CN102140463A - Human miR (microRNA)-1296 antisensenucleic acid and application thereof - Google Patents
Human miR (microRNA)-1296 antisensenucleic acid and application thereof Download PDFInfo
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Abstract
The invention discloses antisense oligonucleotide for suppressing the expression of human miR (microRNA)-1296 and application thereof. The antisense oligonucleotide is specifically combined to human miR-1296, and comprises a sequence complementary to at least 13 continuous nucleotides in a nucleotide sequence, i.e., 5'-UUAGGGCCCUGGCUCCAUCUCC-3', preferably 5'-GGAGAUGGAGCCAGGGCCCUAA-3'. The antisense oligonucleotide disclosed by the invention can be ribonucleotide, deoxyribonucleotide or the chimera of the ribonucleotide and the deoxyribonucleotide, and any ribonucleotide in the chain can be modified. By adopting the miR-1296 antisense oligonucleotide disclosed by the invention, the expression of the miR-1296 in glioma cells of a human brain can be effectively suppressed, the growth and proliferation of the cells are suppressed, and glioma and other tumors with high miR-1296 expression are effectively treated.
Description
Technical field
The present invention relates to biomedicine field.Particularly, the present invention relates to the purposes of a kind of microRNA (miRNA), especially relate to a kind of antisense oligonucleotide and application thereof that people microRNA-1296 (miR-1296) expresses that be used to suppress.This antisense oligonucleotide can with people miR-1296 complementation, thereby suppress the expression of people miR-1296 and play antineoplastic action.The invention still further relates to the pharmaceutical composition that contains this miRNA antisense oligonucleotide.
Background technology
MiRNAs is little non-coding RNA, and length is 20-25bp, is normally transcribed by rna plymerase ii (Pol II), and general initial product is the big pri-miRNA with cap sequence (7MGpppG) and polyadenylic acid tail (AAAAA).These pri-miRNA are processed into the pre-miRNA precursor product that 70 Nucleotide are formed under the effect of RNase III Drosha and its cofactor Pasha.RAN-GTP and exportin 5 are transported to this precursor molecule in the tenuigenin.Subsequently, another RNaseIII Dicer shears it and produces the two strands that is about 22 length of nucleotides.This two strands is directed in (miRISC) complex body very soon, wherein contains Argonaute albumen, and sophisticated strand miRNA is retained in this mixture.Sophisticated miRNA is attached to the site of its complementary mRNA and expresses by two kinds of machine-processed negative regulator genes that depend on the sequence complementarity, suppresses its expression with the incomplete complementary miRNA of said target mrna on the protein translation level.Yet, evidence suggests also that recently these miRNA also might influence the stability of mRNA.Use the miRNA binding site of this mechanism to hold non-translational region at 3 ' of mRNA usually.If miRNA and target site complementary fully (perhaps almost completely complementary), the combination of these miRNA often causes the degraded of target molecule mRNA so.MiRNAs is quite conservative in spore, and animal, the miRNAs that finds in plant and the fungi etc. expresses all strict tissue specificity and sequential.
At present, have only the biological function of very little a part of miRNAs to be illustrated.These miRNAs regulate cell growth and tissue differentiation, and are relevant with biological growth and development.A series of studies show that: miRNAs is at cell growth and apoptosis, and hemocyte breaks up, the homeobox generegulation, and neuronic polarity, insulin secretion, the brain form forms, and heart takes place, and plays a significant role in the processes such as late embryogenesis growth.For example, miR-273 participates in the nervous system development process of nematode; MiR-430 participates in the brain development of zebra fish; MiR-181 control Mammals hematopoietic cell is divided into the B cell; MiR-375 regulates mammalian islet cell and grows and insulin secretion; MiR-143 works in the adipocyte differentiation; MiR-196 has participated in the Mammals four limbs and has formed, and miR-1 is relevant with heart development.Other has the researchist to find that many neural miRNAs are subjected to sequential and regulate in pallium is cultivated, and shows its mRNA that may control compartmentation translation.
MiRNA expresses relevant with multiple cancer, and these genes may play tumor suppressor gene or oncogene effect.In B cell chronic lymphatic leukemia (CLL), find to have the change of miRNA expression level at first, in various human tumors, all detect the miRNA changes of expression level subsequently successively.Discover that it is relevant that miRNAs and tumour form, and can bring into play the effect (as miR-15a and miR-16-1) of tumor suppressor gene, can play the effect (as miR-155 and miR-17-92 bunch) of oncogene again.Think at present, in tumour cell, ripe body of some miRNA or precursor expression horizontal abnormality, and the miRNA of abnormal expression plays a role by influencing the said target mrna translation, participates in neoplastic process, and plays an important role.Be subjected to the regulation and control of let-7 family as the Ras proto-oncogene, the BCL2 anti-apoptotic genes expression is subjected to miR-15a-miR-16-1 bunch of regulation and control, and the E2F1 transcription factor is subjected to miR-17-92 bunch of regulation and control, and the BCL6 anti-apoptotic genes expression is subjected to the regulation and control of miR-127 etc.The down-regulated expression of miRNAs also has substantial connection with tumour, and this is indicating that miRNA has the function of oncogene.For example, miR-143 and miR-145 obviously downward modulation in colorectal carcinoma.What is interesting is that the precursor molecule of its hairpin structure is similar with content in the healthy tissues in tumour, this shows that the down-regulated expression of miRNAs may be because its course of processing is damaged.But the tumor suppressor gene function of miR-143 and miR-145 may not only be confined to colorectal carcinoma, also obviously downward modulation of its expression amount in clones such as mammary cancer, prostate cancer, uterus carcinoma, lymphatic cancer.Another report shows that miR-21 expresses increase in glioblastoma multiforme.The expression amount of this gene in tumor tissues than high 5-100 in healthy tissues doubly.
MiRNAs is natural antisense acting factor, can regulate and control the several genes relevant with propagation with eukaryote existence.Aspect oncotherapy, the application prospect light of miRNA.Utilizing miRNA as aspect the treatment target spot, existing experimental data support:, the variation of miRNA express spectra occurs as in the process of gemcitabine (gemcitabine) treatment; The expression level (as making miR-21 cross expression) of regulation and control part miRNA can be promoted the susceptibility of cholangiocarcinoma cell to chemotherapeutics.MiRNAs by in the possible effectively deactivation tumour of introducing and the miRNA complementary synthetic antisense oligonucleotide---anti-miRNA oligonucleotide (AMOs)---with oncogene characteristic delays its growth.Clinically, can methylate or lock nucleic acid modified antisense oligonucleotide administrations such as (LNA) by 2 '-O-frequent or that continue and make the miRNA inactivation.These modifications make oligonucleotide more stable, and are lower than other treatment means toxicity.Use antagomirs (with cholesterol link coupled AMOs), can effectively suppress the miRNA activity in Different Organs behind the injection mouse, thereby may become a kind of medicine likely.Opposite, cross and express the miRNAs that those have the tumor suppressor gene effect, as let-7 family, also can be used for the treatment of some specific tumour.
Antisense oligonucleotide (Flanagan WM.Antisense comes of age.Cancer﹠amp; Metastasis Reviews 1998; 17 (2): 169-76) be meant one section can with the Nucleotide of the base complementrity of its target gene.Antisense oligonucleotide can be answered expression of gene by inhibitory phase.
People microRNA-1296 (hsa-mir-1296) is positioned at karyomit(e) No. 10, precursor sequence is ACCUACCUAACUGGGUUAGGGCCCUGGCUCCAUCUCCUUUAGGAAAACCUUCUGUG GGGAGUGGGGCUUCGACCCUAACCCAGGUGGGCUGU, contains a ripe microRNA:hsa-miR-1296 (sequence UUAGGGCCCUGGCUCCAUCUCC).
Nearly 30 years, although the complex therapy of tumour is very general clinically, but based on operation, chemicotherapy is that the complex therapy of assisting is also not obvious to the survival rate raising of tumour patient, overall survival rate was still lower in 5 years, paced up and down about 30%～55%, did not significantly improve, 5 years survival rates of middle and advanced stage patient are lower, are about 20%.And all there are limitation separately in these methods, particularly to middle and advanced stage and recurrence patient unsatisfactory curative effect, to poorer with distant metastasis person's curative effect.Therefore, seeking safer and more effective treatment approach is the difficult problem that raising tumour patient survival rate and life quality are needed to be resolved hurrily.
Summary of the invention
The subject matter that the present invention will solve just provides the antisense nucleic acid (inhibitor) of a kind of new miR-1296, be used for efficient, low toxicity or suppress the expression of miR-1296 innocuously, and then treatment and miR-1296 overexpression diseases associated, comprise various noumenal tumours, various leukemia etc.
Another problem that the present invention will solve just provides the purposes of above-mentioned antisense nucleic acid in the medicine of the relative disease (especially cerebral glioma) of preparation treatment miR-1296 overexpression.
The problem again that the present invention will solve provides a kind of pharmaceutical composition that comprises above-mentioned antisense nucleic acid.
The inventor has designed and synthesized the antisense nucleic acid of a species specificity at miR-1296 by extensive and deep research, and checking has the antisense nucleic acid that suppresses effect in culturing cell.Studies show that these antisense nucleic acides can suppress the growth and the malignant proliferation ability of tumour cell.
The present invention has designed a kind of antisense nucleic acid molecule that can specificity be incorporated into miR-1296, in culturing cell U87/MG, the antisense nucleic acid cell growth ability that checking suppresses the miR-1296 expression specificity, the influence of multiplication capacity, antisense nucleic acid molecule length can comprise 13～22 nucleotide residues, inhibition growth of human tumor cells ability in various degree, the characteristic of multiplication capacity are all arranged, wherein the shortest antisense nucleic acid length is 13 bases, and the antisense nucleic acid of different lengths all has good growth of tumour cell and proliferation inhibition activity.Therefore, above-mentioned antisense nucleic acid all can be used to prepare the preparation that suppresses growth of tumour cell ability, multiplication capacity, the wherein tumour cell of preferred miR-1296 high expression level.Finished the present invention on this basis.
A first aspect of the present invention provides the antisense oligonucleotide of a kind of miR-1296, and described antisense oligonucleotide suppresses the expression of miR-1296 in people's cell.Usually, described antisense oligonucleotide and 5 '-UUAGGGCCCUGGCUCCAUCUCC-3 ' in continuous 13～22 nucleotide sequence complementations.In a preferred embodiment of the invention, the length of described antisense oligonucleotide is 18～22 Nucleotide.More preferably, the sequence of described antisense oligonucleotide be 5 '-GGAGAUGGAGCCAGGGCCCUAA-3 '.
At present, the hybridization avidity of RNA and miRNA has very high pharmaceutical use than the avidity height of DNA and miRNA hybridization in the nucleic acid hybridization.But artificial-synthetic DNA's the cost cost than synthetic RNA far away is low, also has good market potential.And can adopt the chimeric antisense nucleic acid that forms that links to each other of ribose RNA monomer and ribodesose dna single body to develop as medicine.A series of antisense nucleic acid molecules of the present invention's design had both comprised dna molecular, also comprised the RNA molecule, and two kinds of molecules all have the activity that suppresses the miR-1296 expression.
The antisense nucleic acid of the present invention's design, it is active that its sequence has specific biological, its antisense nucleic acid institute complementary length for the site of a certain gene complementation has much relations, long as complementary, then biologic activity can be higher, suppressing effect also can be more better, increasing or reducing one to several bases and antisense nucleic acid complementary and same gene locus, equally also has biologic activity in various degree, also can reach in various degree the inhibition growth of tumour cell and the effect of propagation, of the present invention studies show that the shortlyest reaches 13 bases and still has the effect that miR-1296 expresses that suppresses.In the antisense nucleic acid research, various chemical modification methods are a lot.The present invention adopts the one or more combination modified antisense nucleic acid that is selected from ribose modification, base modification and the phosphoric acid backbone modification, preclinical studies such as the pharmacology of the antisense nucleic acid of sulfo-, methoxy modification mode, pharmacokinetics, toxicology are that research is the most comprehensive in the various chemically modified antisense nucleic acides, modified antisense nucleic acid can prevent effectively in vivo in the human body that a large amount of exonucleases cut the enzyme of antisense nucleic acid and degrade, thereby avoids antisense nucleic acid to lose due biologic activity.The sulfo-antisense nucleic acid also can excite the activity of RNA enzyme in addition, the RNA chain of degraded and its hybridization, and therefore preferred these the two kinds antisense nucleic acides of modifying modes are used in experiment.Will be clear that any modifying method that can increase antisense nucleic acid stability and bioavailability can use, as cholesterol modify, PEG modification etc.Antisense nucleic acid modification preferred of the present invention is selected from one or more in thio-modification, 2 '-methoxyl group modification and the cholesterol modification.Most preferably adopt following modification mode: carry out 2 '-methoxyl group and modify, and two Nucleotide of 5 ' end carry out thio-modification, four Nucleotide of 3 ' end carry out thio-modification and connect cholesterol at 5 ' or 3 ' end.
Above-mentioned antisense nucleic acid of the present invention has the effect that suppresses the miR-1296 expression.When with after above-mentioned antisense nucleic acid transfection is in the cell strain U87/MG that expresses miR-1296, can effectively suppress the growth and the malignant proliferation ability of U87/MG cell.
The present invention also provides a kind of pharmaceutical composition, and it contains oligonucleotide of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier includes but not limited to: salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.
Described " significant quantity " is meant and can produces function or amount active and that can be accepted by people and/or animal to people and/or animal.
Described " pharmaceutically acceptable " composition is applicable to people and/or animal and does not have excessive bad side reaction (as toxicity, stimulation and transformation reactions), the material of rational benefit/risk ratio is promptly arranged.
In a third aspect of the present invention, the purposes of antisense oligonucleotide of the present invention is provided, be used to prepare the medicine for the treatment of following disease: treatment human body and miR-1296 cross the expression diseases associated, comprise various noumenal tumours, various leukemia etc.
As used herein, " antisense oligonucleotide " refers to the nucleotide oligomer of antisense.Antisense oligonucleotide by base complementrity (G-C) pairing forms three chains (anti-gene) with double-stranded DNA for A-T, A-U, or forms heteroduplex (antisense) with single stranded RNA, thus the processing and the translation of duplicating, transcribing or transcribing back mRNA of blocking gene.Simultaneously, double-stranded RNA can be degraded by intracellular ribonuclease H (RNaseH), thereby more effectively blocks target gene expression.Because antisense nucleotide can only combine with the target sequence of reverse complemental, has the specificity height, the characteristics that side effect is little.
The length of antisense oligonucleotide of the present invention is not particularly limited, and in general, in order to reach the specificity of hybridization, antisense oligonucleotide need be known the Nucleotide that 13 monomers are formed.Usually the length of antisense oligonucleotide is 13～35bp, and for miRNA, that preferable is 18～24bp.
Description of drawings
Fig. 1 has shown that the miR-1296 antisense oligonucleotide suppresses the expression of miR-1296 in the tumour cell U87/MG cell, and square, circle, trilateral lines are results of revision test among accompanying drawing 1A～D.Figure 1A is the expression of transfection miR-1296 antisense oligonucleotide (5 '-GGAGAUGGAGCCAGGGCCCUAA-3 ') back miR-1296, and Figure 1B be transfection negative control antisense oligonucleotide (5 '-CAGUACUUUUGUGUAGUACAA-3 ') expression of miR-1296 afterwards; Fig. 1 C is the expression of internal control gene U6 behind the transfection miR-1296 antisense oligonucleotide; Fig. 1 D is the expression of internal control gene U6 behind the transfection negative control antisense oligonucleotide; Fig. 1 E is that miR-1296 suppresses the effect histogram behind the transfection antisense oligonucleotide, wherein transfection antisense nucleic acid sample is represented the miR-1296 expression behind the transfection miR-1296 antisense oligonucleotide, and the negative control sample is represented miR-1296 expression behind the transfection negative control.
Embodiment
Antisense oligonucleotide of the present invention, continuous 13～24 nucleotide sequence complementations among its sequence and 5 '-UUAGGGCCCUGGCUCCAUCUCC-3 ', and also not with the RNA sequence complementation of other genes.In a preferred embodiment of the invention, the sequence of described antisense oligonucleotide be 5 '-GGAGAUGGAGCCAGGGCCCUAA-3 '.Antisense oligonucleotide provided by the invention can be modified outcome, it contains at least two, and at least 4 usually, preferable at least 6, at least 8 better Nucleotide do not have the Nucleotide of the modification of toxic side effects, and described modification mode comprises 2 ' methoxyl group replacement, thio-modification etc.In order to increase the cellular uptake rate of antisense oligonucleotide, can also on the basis of above-mentioned modification, carry out cholesterol and modify or the PEGization modification antisense oligonucleotide.Oligonucleotide after the above-mentioned modification can continue effectively to match with target sequence, and has the longer transformation period in vivo than common not modified Yeast Nucleic Acid or thymus nucleic acid.
The present invention has following advantage:
1, antisense oligonucleotide acts on specific target site, the site of non-specific binding seldom, the specificity height;
2, antisense oligonucleotide provided by the invention is through suitable chemically modified, has that toxicity is low, side effect is little and characteristics such as long half time;
3, antisense oligonucleotide provided by the invention has the good restraining effect, and the inhibiting rate of the expression of miR-1296 is reached 40%, and the inhibiting rate of growth of tumour cell is surpassed 60%.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is described in further detail.Yet should be appreciated that and enumerate these embodiment, and be not to be used for limiting the scope of the invention just for an illustration.
At first, by the synthetic miR-1296 antisense nucleic acid of Shanghai JiMa pharmacy Technology Co., Ltd, sequence is: 5 '-GGAGAUGGAGCCAGGGCCCUAA-3 '.The used sequence that relates in an embodiment is synthetic by Shanghai JiMa pharmacy Technology Co., Ltd.
Carry out real-time quantitative fluorescence and detect, the oligonucleotide sequence that wherein relates to is synthetic by Shanghai JiMa pharmacy Technology Co., Ltd, and concrete experimental procedure comprises:
Cell cultures: U87/MG cell (available from typical case's culture collection council of Chinese Academy of Sciences cell bank), 10%FBS-DMEM substratum (FBS is available from Hyclone, and DMEM is available from Gibco) is cultivated, and 37 ℃, 5%CO
2Cultivate.
Cell transfecting:
1) transfection the day before yesterday, with not containing antibiotic culture medium inoculated culturing cell in right amount, the degree of converging of cell reaches 30～50% when making transfection in 24 orifice plates;
2) the transfection sample is prepared oligomer-Lipofecta mine as follows
TM2000 mixtures:
A. the Opti-MEM that does not contain serum with 50 μ l
I substratum (Gibco) dilutes the negative control of miR-1296 antisense oligonucleotide (5 '-GGAGAUGGAGCCAGGGCCCUAA-3 '), negative control (5 '-CAGUACUUUUGUGUAGUACAA-3 '), FAM mark respectively, final concentration is 50nM, mixing gently, each transfection are established 3 multiple holes;
B. mixing Lipofecta mine gently before using
TM2000 (Invitrogen) get the Opti-MEM that 2 μ l are diluted to 50 μ l then
In the I substratum, at room temperature hatch 5min gently behind the mixing;
C. after hatching 5min, the Lipofecta mine of dilution
TM2000 mix with the antisense nucleotide and the contrast of dilution respectively, at room temperature hatch 20min gently behind the mixing, to allow the formation of mixture;
3) mixture is joined in each hole that comprises cell and substratum, the culture plate that rocks back and forth lightly mixes;
4) 37 ℃, 5%CO
2The incubator overnight incubation is changed the substratum that contains 10% foetal calf serum and is continued to cultivate 24h.
Total RNA extracts:
1) centrifugal collecting cell adds 500 μ l Ezol (Invitrogen) in the centrifuge tube, and with the centrifuge tube mixing that turns upside down, room temperature is placed 10min.
2) trichloromethane of adding 200 μ l RNA special uses (worker is given birth in Shanghai), the mixing that acutely turns upside down, the thorough mixing of the liquid in centrifuge tube becomes the oyster white shape.
3) room temperature is placed 5min, the centrifugal 15min of 12000rpm.
4) carefully supernatant is transferred in another clean 1.5ml centrifuge tube, avoids inhaling middle level albumen phase and lower floor's organic phase.
5) add the Virahol (worker is given birth in Shanghai) of the RNA special use of 500 μ l precoolings in the supernatant, room temperature is placed 5min.The centrifugal 10min of 10000rpm.
6) carefully abandon most supernatant, add 75% ethanol (worker is given birth in Shanghai) washing precipitation of 1ml RNA special use, the centrifugal 10min of 10000rpm.
7) carefully abandon most supernatant, place room temperature to dry ethanol, every pipe adds 20 μ l DEPC water (worker is given birth in Shanghai) dissolving, mixing.
The RNA reverse transcription:
The RNA that above-mentioned extracting is obtained carries out reverse transcription with U6 and two kinds of special reverse transcriptase primers of RNA of hsa-miR-1296 respectively, preparation cDNA template.Damping fluid and enzyme used in the reverse transcription are Promega company product.
(i). reverse transcription system (primer sequence is synthetic by Shanghai JiMa pharmacy Technology Co., Ltd):
| Reagent name | Consumption/ |
| 5 * reverse transcription damping fluid | 4μl |
| RT?Primer?Mix (U6，hsa-miR-1296)(1μM) | 1.25μl |
| dNTP(10mM) | 0.75μl |
| RNA | 2μl |
| RTase(200/μl) | 0.5μl |
| DEPC?H 2O | To 20 μ l |
(ii). the reverse transcription reaction condition:
Reaction conditions is: 16 ℃ of 30min; 85 ℃ of lOmin of 42 ℃ of 30min.
Fluorescence quantitative PCR detection:
(1) dilution of .cDNA template:
The cDNA that obtains behind the above-mentioned reverse transcription is diluted 3 times, in the system of 20 μ l, add the ddH2O that 40 μ l do not have RNase/DNase, mixing.
(2). quantitative fluorescent PCR system (primer sequence is synthetic by Shanghai JiMa pharmacy Technology Co., Ltd's design):
| Reagent name | Consumption/pipe |
| 2×PCR?Master?Mix | 10μl |
| F?Primer(20μM) | 0.2μl |
| R?Primer(20μM) | 0.2μl |
| Template | 2μl |
| RTaq archaeal dna polymerase (5U/ul) | 0.2μl |
| ddH2O | Add to 20 μ l |
(3). reaction conditions:
(i)95℃3min；
(ii)95℃15s；
(iii)55℃30s；
(iv)72℃30s；
Ii to the iv goes on foot totally 40 circulations.
Fluorescence quantitative PCR detection result shows that antisense oligonucleotide has the good restraining effect.Accompanying drawing 1 has shown that the miR-1296 antisense oligonucleotide suppresses the expression of miR-1296 in the tumour cell U87/MG cell, and square, circle, trilateral lines are results of revision test among accompanying drawing 1A～D.A is the expression of transfection miR-1296 antisense oligonucleotide (5 '-GGAGAUGGAGCCAGGGCCCUAA-3 ') back miR-1296, and B is the expression of transfection negative control antisense oligonucleotide (5 '-CAGUACUUUUGUGUAGUACAA-3 ') back miR-1296; C is the expression of internal control gene U6 behind the transfection miR-1296 antisense oligonucleotide; D is the expression of internal control gene U6 behind the transfection negative control antisense oligonucleotide; E is that miR-1296 suppresses the effect histogram behind the transfection antisense oligonucleotide, wherein transfection antisense nucleic acid sample is represented the miR-1296 expression behind the transfection miR-1296 antisense oligonucleotide, and the negative control sample is represented miR-1296 expression behind the transfection negative control.
The result shows that antisense oligonucleotide has the obvious suppression effect to the expression of miR-1296.
Embodiment 2, miR-1296 antisense oligonucleotide suppress active to human neuroglia glucagonoma clone U87/MG and detect
The oligonucleotide sequence that wherein relates to is synthetic by Shanghai JiMa pharmacy Technology Co., Ltd.
Cell cultures:
U87/MG cell (available from typical case's culture collection council of Chinese Academy of Sciences cell bank), 10%FBS-DMEM substratum (FBS is available from Gibco, and DMEM is available from Hyclone) is cultivated, and 37 ℃, 5%CO
2Cultivate.Collect the good U87/MG cell of growth conditions, centrifugal counting is with 2 * 10
3Every hole is laid in 96 orifice plates, and 37 ℃, 5%CO
2Cultivate 24h.
Transfection:
1) transfection the day before yesterday, with not containing antibiotic culture medium inoculated culturing cell in right amount, the degree of converging of cell reaches 30～50% when making transfection in 96 orifice plates;
2) the transfection sample is prepared oligomer-Lipofecta mine as follows
TM2000 mixtures:
A. the Opti-MEM that does not contain serum with 25 μ l
I substratum (Gibco) dilutes the negative control of miR-1296 antisense oligonucleotide (5 '-GGAGAUGGAGCCAGGGCCCUAA-3 '), negative control (5 '-CAGUACUUUUGUGUAGUACAA-3 '), FAM mark respectively, final concentration is 50nM after adding in the hand-hole, mixing gently, each transfection are established 3 multiple holes;
B. mixing Lipofecta mine gently before using
TM2000 (Invitrogen) get the Opti-MEM that 0.25 μ l is diluted to 25 μ l then
The I substratum is at room temperature hatched 5min gently behind the mixing;
C. after hatching 5min, the Lipofecta mine of dilution
TM2000 mix with the antisense nucleotide and the contrast of dilution respectively, at room temperature hatch 20min gently behind the mixing, to allow the formation of mixture;
3) mixture is joined in each hole that comprises cell and substratum, the culture plate that rocks back and forth lightly mixes; The final concentration of antisense nucleotide and contrast is 50nM.
4) 37 ℃, 5%CO
2After incubator continued to hatch 72 hours, microscopic examination U87/MG cell was taken a picture.
Cytotoxicity experiment based on MTT:
Upwards the cell that obtains in the step adds MTT (Sigma) 5mg/ml (the physiological saline preparation with 0.9%) for preparing, and every hole adds 20 μ l, 37 ℃, 5%CO
2Hatch to inhale after 4 hours and remove substratum and MTT, every hole adds DMSO 100 μ l and reads the absorbance of OD570-OD630 by microplate reader.
Calculate inhibiting rate:
Calculating inhibiting rate is 72.21 ± 3.31%.
The result shows: miR-1296 antisense oligonucleotide provided by the invention has the good restraining effect, and the inhibiting rate that U87/MG is grown surpasses 70%.
Claims (10)
1. an antisense oligonucleotide is characterized in that, described antisense oligonucleotide comprise with 5 '-UUAGGGCCCUGGCUCCAUCUCC-3 ' in continuous 13～22 nucleotide sequence complementary sequences.
2. antisense oligonucleotide as claimed in claim 1 is characterized in that described antisense oligonucleotide comprises sequence 5 '-GGAGAUGGAGCCAGGGCCCUAA-3 '.
3. antisense oligonucleotide as claimed in claim 1 is characterized in that, described antisense oligonucleotide is the mosaic of ribonucleotide, deoxyribonucleotide or ribonucleotide and deoxyribonucleotide.
4. as each described antisense oligonucleotide in the claim 1～3, it is characterized in that described antisense oligonucleotide is further modified.
5. antisense oligonucleotide as claimed in claim 4 is characterized in that, described modification is selected from one or more the combination in ribose modification, base modification and the phosphoric acid backbone modification.
6. antisense oligonucleotide as claimed in claim 5 is characterized in that, described modification is selected from one or more in thio-modification, 2 '-methoxyl group modification and the cholesterol modification.
7. antisense oligonucleotide as claimed in claim 6, it is characterized in that, all nucleic acid carry out 2 '-methoxyl group to be modified, and two Nucleotide of 5 ' end carry out thio-modification, and four Nucleotide of 3 ' end carry out thio-modification and connect cholesterol at 5 ' or 3 ' end.
8. each described antisense oligonucleotide is used to prepare the purposes of medicine of the relative disease of treatment miR-1296 overexpression in the claim 1～7.
9. purposes as claimed in claim 8 is characterized in that, the relative disease of described miR-1296 overexpression is a cerebral glioma.
10. a pharmaceutical composition is characterized in that, contains each described antisense oligonucleotide and pharmaceutically acceptable carrier in the claim 1～7 for the treatment of significant quantity.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101091798A (en) * | 2007-04-17 | 2007-12-26 | 华东师范大学 | Application of meaning inversed miRNA-210 |
| CN101535331A (en) * | 2005-04-29 | 2009-09-16 | 洛克菲勒大学 | Human micrornas and methods for inhibiting same |
| WO2009149182A1 (en) * | 2008-06-04 | 2009-12-10 | The Board Of Regents Of The University Of Texas System | Modulation of gene expression through endogenous small rna targeting of gene promoters |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101535331A (en) * | 2005-04-29 | 2009-09-16 | 洛克菲勒大学 | Human micrornas and methods for inhibiting same |
| CN101091798A (en) * | 2007-04-17 | 2007-12-26 | 华东师范大学 | Application of meaning inversed miRNA-210 |
| WO2009149182A1 (en) * | 2008-06-04 | 2009-12-10 | The Board Of Regents Of The University Of Texas System | Modulation of gene expression through endogenous small rna targeting of gene promoters |
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