CN102140462B - Human miR-1260 antisense nucleic acid and application thereof - Google Patents
Human miR-1260 antisense nucleic acid and application thereof Download PDFInfo
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Abstract
The invention discloses antisense oligonucleotide inhibiting human microRNA-1260 from expressing and application thereof. The antisense oligonucleotide is specifically combined with human miR-1260, contains a sequence complementary with at least 13 continuous nucleotides in a 5'-AUCCCACCUCUGCCACCA-3' nucleotide sequence, particularly a sequence of 5'-UGGUGGCAGAGGUGGGAU-3'. The antisense oligonucleotide can be ribonucleotide, deoxyribonucleotide or chimera of both, and can modify any nucleotide in a chain. The miR-1260 antisense oligonucleotide effectively inhibits miR-1260 in a human glioma cell from expressing, and inhibits the cell from growing and propagating so as to effectively treat human glioma and other miR-1260 high-expression tumors.
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 for suppressing people microRNA-1260 (miR-1260) expression.This antisense oligonucleotide can be complementary with people miR-1260, thereby suppress the expression of people miR-1260 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 (PolII), and general initial product is the large 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 form under the effect of RNase III Drosha and its cofactor Pasha.RAN-GTP and exportin 5 are transported to this precursor molecule in 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 ripe strand miRNA is retained in this mixture.Ripe miRNA is attached to the site of the mRNA complementary with it and expresses by two kinds of machine-processed negative regulator genes that depend on the sequence complementarity, and the miRNA not exclusively complementary with said target mrna suppresses its expression on the protein translation level.Yet, also evidence suggests recently, these miRNA also might affect the stability of mRNA.Use the miRNA binding site of this mechanism usually to hold non-translational region at 3 ' of mRNA.If miRNA and target site complete complementary (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 fungi etc. expresses all strict tissue specificity and timing.
At present, only have the biological function of very little a part of miRNAs to be elucidated.These miRNAs regulate Growth of Cells and tissue differentiation, and are relevant with biological growth and development.A series of studies show that: miRNAs is at Growth of Cells and apoptosis, and hemocyte breaks up, and Homeobox gene is regulated, neuronic polarity, and insulin secretion, the brain form forms, and heart occurs, 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 controls the Mammals hematopoietic cell and is divided into the B cell; MiR-375 regulates mammalian islet cell and grows and insulin secretion; MiR-143 works at Adipocyte Differentiation; MiR-196 has participated in the Mammals four limbs and has formed, and miR-1 is relevant with heart development.Separately there is the researchist to find that many neural miRNAs are subject to sequential and regulate in pallium is cultivated, shows the mRNA translation that it may control compartmentation.
MiRNA expresses relevant to kinds cancer, and these genes may play tumor suppressor gene or oncogene effect.Find to have at first the change of miRNA expression level in B cell chronic lymphatic leukemia (CLL), the variation of miRNA expression level all detected successively subsequently in various human tumors.Research finds, 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 again the effect (as miR-155 and miR-17-92 bunch) of oncogene.Think at present, in tumour cell, the ripe body of some miRNA or precursor expression horizontal abnormality, and the miRNA of abnormal expression plays a role by affecting 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 lower in colorectal carcinoma.What is interesting is, the precursor molecule of its hairpin structure is similar with content in healthy tissues in tumour, and this shows, 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, and its expression amount is also obviously lowered in the clones such as mammary cancer, prostate cancer, uterus carcinoma, lymphatic cancer.Another report shows, 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 to eukaryote existence.Aspect oncotherapy, the application prospect of miRNA is bright.Utilizing miRNA as aspect the treatment target spot, existing experimental data support: as in the process of gemcitabine (gemcitabine) treatment, the variation of miRNA express spectra occurs; The expression level (as making miR-21 cross expression) of regulation and control part miRNA can be promoted cholangiocarcinoma cell to the susceptibility of chemotherapeutics.By introducing and the effective miRNAs in the deactivation tumour of the synthetic antisense oligonucleotide---anti-miRNA oligonucleotide (AMOs)---of the miRNA complementation with oncogene characteristic, delay its growth.Clinically, can methylate or the antisense oligonucleotide administration of locking the modifications such as nucleic acid (LNA) makes the miRNA inactivation by 2 '-O-frequent or that continue.These modifications make oligonucleotide more stable, and are lower than other treatment means toxicity.Use antagomirs (with the AMOs of cholesterol coupling), can effectively suppress the miRNA activity in Different Organs after 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) refer to one section can with the Nucleotide of the base complementrity of its target gene.Antisense oligonucleotide can suppress the expression of corresponding gene.
People microRNA-1260 (hsa-mir-1260) is positioned at karyomit(e) No. 14, precursor sequence is ACCUUUCCAGCUCAUCCCACCUCUGCCACCAAAACACUCAUCGCGGGGUCAGAGGG AGUGCCAAAAAAGGUAA, contains a ripe microRNA:hsa-miR-1260 (sequence is AUCCCACCUCUGCCACCA).
Nearly 30 years, although the complex therapy of tumour is very general clinically, but take operation as main, to be auxiliary complex therapy improve and not obvious the survival rate of tumour patient chemicotherapy, overall survival rate was still lower in 5 years, hovered in 30%～55% left and right, 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 patients with recurrent 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 need to be resolved hurrily.
Summary of the invention
The subject matter that the present invention will solve just is to provide the antisense nucleic acid (inhibitor) of a kind of new miR-1260, be used for efficient, low toxicity or suppress innocuously the expression of miR-1260, and then the treatment disease relevant with the miR-1260 overexpression, comprise various noumenal tumours, various leukemia etc.
Another problem that the present invention will solve just is to provide the purposes of above-mentioned antisense nucleic acid in the medicine of the relative disease (especially cerebral glioma) of preparation treatment miR-1260 overexpression.
The problem again that the present invention will solve is to provide 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 for miR-1260 by extensive and deep research, and the antisense nucleic acid that checking has inhibition in culturing cell.Studies show that growth and malignant proliferation ability that these antisense nucleic acides can inhibition tumor cell.
The present invention designed a kind of can specific binding in the antisense nucleic acid molecule of miR-1260, in culturing cell U87/MG, the antisense nucleic acid cell growth ability that checking suppresses the miR-1260 expression specificity, the impact of multiplication capacity, antisense nucleic acid molecule length can comprise 13～24 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 of inhibition tumor cell energy for growth, multiplication capacity, wherein the tumour cell of preferred miR-1260 high expression level.Completed on this basis the present invention.
A first aspect of the present invention provides the antisense oligonucleotide of a kind of miR-1260, and described antisense oligonucleotide suppresses the expression of miR-1260 in people's cell.Usually, described antisense oligonucleotide and 5 '-AUCCCACCUCUGCCACCA-3 ' in continuous 13～18 nucleotide sequence complementations.In a preferred embodiment of the invention, the length of described antisense oligonucleotide is 13～18 Nucleotide.More preferably, the sequence of described antisense oligonucleotide be 5 '-UGGUGGCAGAGGUGGGAU-3 '.
At present, in nucleic acid hybridization, RNA is higher than the avidity of DNA and miRNA hybridization with the hybridization avidity of miRNA, has very high pharmaceutical use.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 is connected 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-1260 expression.
the antisense nucleic acid of the present invention's design, it is active that its sequence has specific biological, it has much relations for the complementary length of the antisense nucleic acid in the site of a certain gene complementation, length as complementation is a little, biologic activity can be higher, inhibition also can be more better, increasing or reducing one to several bases and antisense nucleic acid complementary and the same gene site, equally also has biologic activity in various degree, also can reach in various degree inhibition tumor cell growth and the effect of breeding, of the present invention studies show that, the shortlyest reach 13 bases and still have the effect that miR-1260 expresses that suppresses.In antisense nucleic acid research, various chemical modification methods are a lot.The present invention adopts the antisense nucleic acid of the one or more combination modification that is selected from ribose modification, base modification and phosphoric acid backbone modification, the preclinical studies such as the pharmacology of the antisense nucleic acid of sulfo-, methoxy modification mode, pharmacokinetics, toxicology are that in various chemically modified antisense nucleic acides, research is the most comprehensive, the antisense nucleic acid of modifying can prevent effectively that in vivo in human body, a large amount of exonucleases are cut and degrade the enzyme of antisense nucleic acid, 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 that degraded is hybrid with it, and therefore preferred these the two kinds antisense nucleic acides of modifying mode 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 preferred of the present invention is modified one or more that are selected from thio-modification, 2 '-methoxyl group modification and 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-1260 expression.After in above-mentioned antisense nucleic acid being transfected into the cell strain U87/MG that expresses miR-1260, can effectively suppress 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 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 " refers to and can produce 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 without excessive bad side reaction (as toxicity, stimulation and transformation reactions), the material of rational benefit/risk ratio is arranged namely.
In a third aspect of the present invention, the purposes of antisense oligonucleotide of the present invention is provided, for the preparation of the medicine of the following disease for the treatment of: treatment human body and miR-1260 cross the relevant disease of expression, comprise various noumenal tumours, various leukemia etc.
As used herein, " antisense oligonucleotide " refers to the nucleotide oligomer of antisense.Antisense oligonucleotide is by base complementrity (A-T, A-U, G-C) pairing forms three chains (anti-gene) with double-stranded DNA, or forms heteroduplex (antisense) with single stranded RNA, thus the processing and the translation that copy, transcribe or transcribe rear mRNA of blocking gene.Simultaneously, double-stranded RNA can be degraded by intracellular ribonuclease H (RNaseH), thereby more effectively blocks the expression of target gene.Because antisense nucleotide can only be combined with the target sequence of reverse complemental, has specificity high, 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 needs the Nucleotide of at least 13 monomer compositions.Usually the length of antisense oligonucleotide is 13～35bp, and for miRNA, that better is 18～24bp.
Description of drawings
Fig. 1 has shown the expression of miR-1260 in miR-1260 antisense oligonucleotide inhibition tumor cell U87/MG cell, and in accompanying drawing 1A～D, square, circle, trilateral lines are results of revision test.A is the expression of miR-125a-5p after transfection miR-1260 antisense oligonucleotide (5 '-UGGUGGCAGAGGUGGGAU-3 '), and B is the expression of miR-1260 after transfection negative control antisense oligonucleotide (5 '-CAGUACUUUUGUGUAGUACAA-3 '); C is the expression of reference gene U6 after transfection miR-1260 antisense oligonucleotide; D is the expression of reference gene U6 after transfection negative control antisense oligonucleotide; E is miR-1260 inhibition histogram after the transfection antisense oligonucleotide, wherein transfection antisense nucleic acid sample represents the miR-1260 expression after transfection miR-1260 antisense oligonucleotide, and the negative control sample represents miR-1260 expression after the transfection negative control.
Embodiment
Antisense oligonucleotide of the present invention, continuous 13～18 nucleotide sequence complementations in its sequence and 5 '-AUCCCACCUCUGCCACCA-3 ', and also not complementary with the RNA sequence of other genes.In a preferred embodiment of the invention, the sequence of described antisense oligonucleotide be 5 '-UGGUGGCAGAGGUGGGAU-3 '.Antisense oligonucleotide provided by the invention can be modified outcome, it contains at least two, and at least 4 usually, better 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 ' methoxy substitution, thio-modification etc.In order to increase the cellular uptake rate of antisense oligonucleotide, can also carry out cholesterol to antisense oligonucleotide on the basis of above-mentioned modification and modify or the PEGization modification.Oligonucleotide after above-mentioned modification can continue effectively to match with target sequence, and has in vivo the longer transformation period 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, specificity is high;
2, antisense oligonucleotide provided by the invention is through suitable chemically modified, has that toxicity is low, side effect is little and the characteristics such as long half time;
3, antisense oligonucleotide provided by the invention has good inhibition, and the inhibiting rate of the expression of miR-1260 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 just for an illustration, and be not to limit the scope of the invention.
At first, by the synthetic miR-1260 antisense nucleic acid of Shanghai JiMa pharmacy Technology Co., Ltd, sequence is: 5 '-UGGUGGCAGAGGUGGGAU-3 '.The sequence used 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 in right amount antibiotic culture medium inoculated culturing cell, when making transfection, the degree of converging of cell reaches 30～50% in 24 orifice plates;
2) the transfection sample is prepared oligomer-Lipofecta mine as follows
TM2000 mixtures:
A. do not contain the Opti-MEM of serum with 50 μ l
I substratum (Gibco) dilutes respectively the negative control of miR-1260 antisense oligonucleotide (5 '-UGGUGGCAGAGGUGGGAU-3 '), negative control (5 '-CAGUACUUUUGUGUAGUACAA-3 '), FAM mark, final concentration is 50nM, mixing gently, each transfection are established 3 multiple holes;
B. mixing Lipofecta mine gently before using
TM2000 (Invitrogen) then get the Opti-MEM that 2 μ l are diluted to 50 μ l
In the I substratum, at room temperature hatch gently 5min after mixing;
C. after hatching 5min, the Lipofecta mine of dilution
TM2000 mix with antisense nucleotide and the contrast of dilution respectively, at room temperature hatch gently 20min after mixing, to allow complex formation;
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 centrifuge tube, with the centrifuge tube mixing that turns upside down, room temperature is placed 10min.
2) add the trichloromethane (work is given birth in Shanghai) of 200 μ l RNA special uses, the mixing that acutely turns upside down is until 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 (work 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 (work is given birth in the Shanghai) washing precipitation of 1ml RNA special use, the centrifugal 10min of 10000rpm.
7) carefully abandon most supernatant, be placed in room temperature and dry ethanol, every pipe adds 20 μ l DEPC water (work 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-1260 respectively, preparation cDNA template.In reverse transcription, damping fluid used and enzyme are Promega company product.
(i). reverse transcription system (primer sequence is synthetic by Shanghai JiMa pharmacy Technology Co., Ltd):
| Reagent name | Consumption/pipe |
| 5 * reverse transcription damping fluid | 4μl |
| RT Primer Mix (U6，hsa-miR-1260)(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; 42 ℃ of 30min; 85 ℃ of 10min.
Fluorescence quantitative PCR detection:
(1) dilution of .cDNA template:
The cDNA that obtains after above-mentioned reverse transcription is diluted 3 times, add 40 μ l without the ddH2O of RNase/DNase, mixing in the system of 20 μ l.
(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.
The fluorescence quantitative PCR detection result shows that antisense oligonucleotide has good inhibition.Accompanying drawing 1 has shown the expression of miR-1260 in miR-1260 antisense oligonucleotide inhibition tumor cell U87/MG cell, and in accompanying drawing 1A～D, square, circle, trilateral lines are results of revision test.A is the expression of miR-1260 after transfection miR-1260 antisense oligonucleotide (5 '-UGGUGGCAGAGGUGGGAU-3 '), and B is the expression of miR-125a-5p after transfection negative control antisense oligonucleotide (5 '-CAGUACUUUUGUGUAGUACAA-3 '); C is the expression of reference gene U6 after transfection miR-1260 antisense oligonucleotide; D is the expression of reference gene U6 after transfection negative control antisense oligonucleotide; E is miR-1260 inhibition histogram after the transfection antisense oligonucleotide, wherein transfection antisense nucleic acid sample represents the miR-1260 expression after transfection miR-1260 antisense oligonucleotide, and the negative control sample represents miR-1260 expression after the transfection negative control.
Result shows that antisense oligonucleotide has obvious restraining effect to the expression of miR-1260.
Embodiment 2, miR-1260 antisense oligonucleotide are that U87/MG suppresses active detection to the neuroglia cell of human oncocyte
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 in right amount antibiotic culture medium inoculated culturing cell, when making transfection, the degree of converging of cell reaches 30～50% in 96 orifice plates;
2) the transfection sample is prepared oligomer-Lipofecta mine as follows
TM2000 mixtures:
A. do not contain the Opti-MEM of serum with 25 μ l
I substratum (Gibco) dilutes respectively the negative control of miR-1260 antisense oligonucleotide (5 '-UGGUGGCAGAGGUGGGAU-3 '), negative control (5 '-CAGUACUUUUGUGUAGUACAA-3 '), FAM mark, after adding in hand-hole, final concentration is 50nM, mixing gently, each transfection are established 3 multiple holes;
B. mixing Lipofecta mine gently before using
TM2000 (Invitrogen) then get the Opti-MEM that 0.25 μ l is diluted to 25 μ l
The I substratum is at room temperature hatched 5min gently after mixing;
C. after hatching 5min, the Lipofecta mine of dilution
TM2000 mix with antisense nucleotide and the contrast of dilution respectively, at room temperature hatch gently 20min after mixing, to allow complex formation;
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:
The cell that obtains in the previous 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 after 4 hours and suck substratum and MTT, every hole adds DMSO 100 μ l and reads the absorbance of OD570-OD630 by microplate reader.
The sample negative control
(OD570-OD630)(OD570-OD630)
0.185 0.548
0.175 0.421
0.191 0.494
Calculate inhibiting rate:
Calculating inhibiting rate is 62.40 ± 1.62%.
Result shows: miR-1260 antisense oligonucleotide provided by the invention has good inhibition, and the inhibiting rate that U87/MG is grown surpasses 60%.
Sequence table
＜110〉Suzhou JiMa gene medicine Science Co., Ltd
＜120〉people miR-1260 antisense nucleic acid and application thereof
<130>201010164079.3
<160>2
<170>Patentln version 3.3
<210>1
<211>18
<212>RNA
<213>Homo sapiens
<400>1
aucccaccuc ugccacca 18
<210>2
<211>18
<212>DNA
<213>Artificial
<220>
<223>artificial
<400>2
ugguggcaga ggugggau 18
Claims (6)
1. an antisense oligonucleotide is for the preparation of the purposes of the medicine for the treatment of cerebral glioma, wherein, described antisense oligonucleotide sequence is the chimeric sequence with ribonucleoside acid sequence, deoxyribonucleotide sequence or ribonucleotide and the deoxyribonucleotide of the complementation of 5 '-AUCCCACCUCUGCCACCA-3 ' nucleotide sequence.
2. purposes as claimed in claim 1, is characterized in that, the sequence of described antisense oligonucleotide is 5 '-UGGUGGCAGAGGUGGGAU-3 '.
3. purposes as claimed in claim 1 or 2, is characterized in that, described antisense oligonucleotide is further modified.
4. purposes as claimed in claim 3, is characterized in that, described modification is selected from one or more the combination in ribose modification, base modification and phosphoric acid backbone modification.
5. purposes as claimed in claim 4, is characterized in that, described modification is selected from one or more in thio-modification, 2 '-methoxyl group modification and cholesterol modification.
6. purposes as claimed in claim 5, is characterized in that, described being modified to: 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.
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| CN101031657A (en) * | 2004-05-14 | 2007-09-05 | 罗塞塔金诺米克斯有限公司 | Microrna and uses thereof |
| US20090143326A1 (en) * | 2007-10-04 | 2009-06-04 | Santaris Pharma A/S | MICROMIRs |
| CN101622350A (en) * | 2006-12-08 | 2010-01-06 | 奥斯瑞根公司 | miR-126 regulated genes and pathways as targets for therapeutic intervention |
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| CN101031657A (en) * | 2004-05-14 | 2007-09-05 | 罗塞塔金诺米克斯有限公司 | Microrna and uses thereof |
| CN101622350A (en) * | 2006-12-08 | 2010-01-06 | 奥斯瑞根公司 | miR-126 regulated genes and pathways as targets for therapeutic intervention |
| US20090143326A1 (en) * | 2007-10-04 | 2009-06-04 | Santaris Pharma A/S | MICROMIRs |
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