CN102080083B - Human miR-149 antisense nucleotide and application thereof - Google Patents
Human miR-149 antisense nucleotide and application thereof Download PDFInfo
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Abstract
The invention discloses an antisense oligonucleotide for inhibiting human microRNA-149 expression. The antisense oligonucleotide disclosed by the invention comprises sequences which are complementary with 13 to 23 successive nucleotides in 5'-UCUGGCUCCGUGUCUUCACUCCC-3', especially comprising a sequence 5'-GGGAGUGAAGACACGGAGCCAGA-3' which can be specifically combined with the human miR-149. The antisense oligonucleotide provided by the invention can be ribonucleotide, deoxyribonucleotide or a chimera of ribonucleotide and deoxyribonucleotide, and can modify any nucleotide in a chain. The miR-149 antisense nucleotide provided by the invention can effectively inhibit the miR-149 expression in a human brain glioma cell, and inhibit the development and multiplication of the miR-149 antisense nucleotide, thereby effectively treating the brain glioma tumor and other tumors with the miR-149 high expression.
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-149 (miR-149) expression.This antisense oligonucleotide can be complementary with people miR-149, thereby suppress the expression of people miR-149 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, length is 20-25bp,, normally to be transcribed by rna plymerase ii (PolII), 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 the tenuigenin.Subsequently, another RNase III 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 in the protein translation level.Yet, evidence suggests also that 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 the 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 control Mammals hematopoietic cell 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.Other has the researchist to find that many neural miRNAs are subject to sequential and regulate in pallium is cultivated, and shows the mRNA translation that it may control compartmentation.
MiRNA expresses relevant with kinds cancer, and these genes may play tumor suppressor gene or oncogene effect.In B cell chronic lymphatic leukemia (CLL), find to have at first the change of miRNA expression level, in various human tumors, all detect successively subsequently the variation of miRNA expression level.Research finds, it is relevant that miRNAs and tumour form, and can bring into play the effect (such as miR-15a and miR-16-1) of tumor suppressor gene, can play again the effect (such 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 such 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 the 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 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 are so that oligonucleotide is more stable, and are lower than other treatment means toxicity.Use antagomirs (with the AMOs of cholesterol coupling), can be active at Different Organs establishment miRNA 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, also can be used for the treatment of some specific tumour such as let-7 family.
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-149 (hsa-mir-149) is positioned at karyomit(e) No. 2, precursor sequence is GCCGGCGCCCGAGCUCUGGCUCCGUGUCUUCACUCCCGUGCUUGUCCGAGGAGGGA GGGAGGGACGGGGGCUGUGCUGGGGCAGCUGGA, contains two ripe microRNA:hsa-miR-149 (sequence is UCUGGCUCCGUGUCUUCACUCCC) and hsa-miR-149* (sequence is AGGGAGGGACGGGGGCUGUGC).Schaefer etc. have utilized microarray method and the RT-qPCR methods analyst sample of 76 routine radical prostatectomies is found that hsa-miR-149 expresses to descend.The usefulness Taq Man MicroRNA such as Wong have detected miRNA expression level in 20 pairs of squamous cell tongue cancers, find that the hsa-miR-149 expression level is higher 3 times than healthy tissues.But also do not report about the function of mir-149 and the research of expression level in glioma.(Wong，Liu et al.2008；Schaefer，Jung et al.2009)。
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, 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 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 provides the antisense nucleic acid (inhibitor) of a kind of new miR-149, be used for efficient, low toxicity or suppress innocuously the expression of miR-149, and then the treatment disease relevant with the miR-149 overexpression, 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-149 overexpression.
The again problem 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 for miR-149 by extensive and deep research, and checking has the effect that suppresses miR-149 expression and tumor cell proliferation in culturing cell.Studies show that growth and malignant proliferation ability that these antisense nucleic acides can inhibition tumor cell.
The present invention has designed the antisense nucleic acid molecule that can be incorporated into the miR-149 different positions in, in culturing cell U87/MG, and the antisense nucleic acid cell growth ability that checking suppresses the miR-149 expression specificity, the impact of multiplication capacity.Antisense nucleic acid molecule length can comprise 13～23 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-149 high expression level.Finished on this basis the present invention.
A first aspect of the present invention provides the antisense oligonucleotide of a kind of miR-149, and described antisense oligonucleotide suppresses the expression of miR-149 in people's cell.Usually, continuous 13～23 nucleotide sequence complementations among described antisense oligonucleotide and the 5 '-UCUGGCUCCGUGUCUUCACUCCC-3 '.In a preferred embodiment of the invention, the length of described antisense oligonucleotide is 18～23 Nucleotide.More preferably, the sequence of described antisense oligonucleotide be 5 '-GGGAGUGAAGACACGGAGCCAGA-3 '.
At present, RNA is higher than the avidity of DNA and miRNA hybridization with the hybridization avidity of miRNA in the nucleic acid hybridization, has very high pharmaceutical use.But artificial-synthetic DNA's the cost far away cost than synthetic RNA 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-149 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 such as complementation is a little, then 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 reaches 13 bases and still has the effect that miR-149 expresses that suppresses.In the 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 the 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 research is the most comprehensive in the various chemically modified antisense nucleic acides, the antisense nucleic acid of modifying 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 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 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-149 expression.After in above-mentioned antisense nucleic acid being transfected into the cell strain U87/MG that expresses miR-149, growth and malignant proliferation ability that can the establishment tumour 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 (such 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 treatment following disease medicine:
The treatment human body is crossed with miR-149 and is expressed relevant disease, comprises various noumenal tumours, various leukemia etc., especially cerebral glioma.
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 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 to be known the Nucleotide of 13 monomer compositions.Usually the length of antisense oligonucleotide is 13～35bp, and for miRNA, that better is 18～23bp.
Description of drawings
Fig. 1 has shown the expression of miR-149 in the miR-149 antisense oligonucleotide inhibition tumor cell U87/MG cell, and square, circle, trilateral lines are results of revision test among accompanying drawing 1A～D.A is the expression of miR-149 behind the transfection miR-149 antisense oligonucleotide (5 '-GGGAGUGAAGACACGGAGCCAGA-3 '), and B is the expression of miR-149 behind the transfection negative control antisense oligonucleotide (5 '-CAGUACUUUUGUGUAGUACAA-3 '); C is the expression that turns reference gene U6 behind the miR-149 antisense oligonucleotide; D is the expression of reference gene U6 behind the transfection negative control antisense oligonucleotide; E is miR-149 inhibition histogram behind the transfection antisense oligonucleotide, wherein transfection antisense nucleic acid sample represents the miR-149 expression behind the transfection miR-149 antisense oligonucleotide, and the negative control sample represents miR-149 expression behind the transfection negative control.
Fig. 2 has shown growth and the propagation of miR-149 antisense oligonucleotide inhibition tumor cell U87/MG cell, the U87/MG cell behind A, B the have been transfection negative control of FAM mark; C is U87/MG cell state behind the transfection negative control; D is U87/MG cell state behind the transfection miR-149 antisense oligonucleotide (5 '-GGGAGUGAAGACACGGAGCCAGA-3 ').
Embodiment
Antisense oligonucleotide of the present invention, continuous 13～23 nucleotide sequence complementations among its sequence and 5 '-UCUGGCUCCGUGUCUUCACUCCC-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 is 5 '-GGGAGUGAAGACACGGAGCCAGA-3 '.Antisense oligonucleotide provided by the invention is 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 on the basis of above-mentioned modification, carry out cholesterol to antisense oligonucleotide and modify or the PEGization modification.Oligonucleotide after the 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-149 is reached more than 70%, and the inhibiting rate of growth of tumour cell is surpassed 45%.
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-149 antisense nucleic acid of Shanghai JiMa pharmacy Technology Co., Ltd, sequence is: 5 '-GGGAGUGAAGACACGGAGCCAGA-3 '.The used sequence that relates in an embodiment is synthetic by Shanghai JiMa pharmacy Technology Co., Ltd.
Embodiment 1, miR-149 antisense nucleic acid suppress the expression of miR-149
Carry out real-time quantitative fluorescence and detect, the oligonucleotide sequence that wherein relates to and primer sequence are 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, 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. do not contain serum with 50 μ l
I substratum (Gibco) dilutes respectively the negative control of miR-149 antisense oligonucleotide (5 '-GGGAGUGAAGACACGGAGCCAGA-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 2 μ l and are diluted to 50 μ l's
In the I substratum, at room temperature hatch gently 5min behind the 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 behind the 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 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 is until the thorough mixing of the liquid in the 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 dissolution, 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-149 respectively, preparation cDNA template.Damping fluid and enzyme used in the reverse transcription are Promega company product.Mir-149RT primer: 5 '-GTCTGTATGGTTGTTCTGCTCTCTGTCTCATCCCTATCTACAACCATACAGACGGG AGT-3 '
(i). the reverse transcription system:
| Reagent name | Consumption/pipe |
| 5 * reverse transcription damping fluid | 4μl |
| RT Primer Mix (U6，hsa-miR-149)(1μM) | 1.25μl |
| dNTP(10mM) | 0.75μl |
| RNA | 2μl |
| RTase(200/μl) | 0.5μl |
| DEPC H2O | 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 behind the above-mentioned reverse transcription is diluted 3 times, in the system of 20 μ l, add 40 μ l without the ddH2O of RNase/DNase, mixing.
(2). (primer sequence is synthetic by Shanghai JiMa pharmacy Technology Co., Ltd for the quantitative fluorescent PCR system; Mir-149FP primer: 5 '-ATAACTcTggcTccgTgTcTTc-3 '; Mir-149RP primer: 5 '-TATGGTTGTTCTGCTCTCTGTCTC-3 ')
| Reagent name | Consumption/ |
| 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 good inhibition.Accompanying drawing 1 has shown the expression of miR-149 in the miR-149 antisense oligonucleotide inhibition tumor cell U87/MG cell, and square, circle, trilateral lines are results of revision test among accompanying drawing 1A～D.A is the expression of miR-149 behind the transfection miR-149 antisense oligonucleotide (5 '-GGGAGUGAAGACACGGAGCCAGA-3 '), and B is the expression of miR-149 behind the transfection negative control antisense oligonucleotide (5 '-CAGUACUUUUGUGUAGUACAA-3 '); C is the expression that turns reference gene U6 behind the miR-149 antisense oligonucleotide; D is the expression of reference gene U6 behind the transfection negative control antisense oligonucleotide; E is miR-149 inhibition histogram behind the transfection antisense oligonucleotide, wherein transfection antisense nucleic acid sample represents the miR-149 expression behind the transfection miR-149 antisense oligonucleotide, and the negative control sample represents miR-149 expression behind the transfection negative control.The result shows that antisense oligonucleotide has obvious restraining effect to the expression of miR-149.
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, 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. do not contain serum with 25 μ l
I substratum (Gibco) dilutes respectively the negative control of miR-149 antisense oligonucleotide (5 '-GGGAGUGAAGACACGGAGCCAGA-3 '), negative control (5 '-CAGUACUUUUGUGUAGUACAA-3 '), FAM mark, 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) then get 0.25 μ l and are diluted to 25 μ l's
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 antisense nucleotide and the contrast of dilution respectively, at room temperature hatch gently 20min behind the 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.
As shown in Figure 2, transfection is after 72 hours, surpassed 80% U87/MG cell success transfection the negative control of FAM mark (Fig. 2 A, 2B); Behind the transfection negative control, U87/MG cell fraction of coverage is high, and cell is complete, light transmission strong (Fig. 2 C); And most of U87/MG necrocytosis (Fig. 2 D) behind the transfection miR-149 antisense oligonucleotide.
Cytotoxicity experiment based on MTT:
The cell that obtains in the previous step adds MTT (Sigma) 5mg/ml (physiological saline with 0.9% is prepared) 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.
Calculate inhibiting rate:
Calculating inhibiting rate is 42.85 ± 6.63%.The result shows: miR-149 antisense oligonucleotide provided by the invention has good inhibition, to the inhibiting rate of U87/MG growth near 45%.
Sequence table
＜110〉Shanghai Pharmaceutical Inst., Chinese Academy of Sciences
Suzhou JiMa gene medicine Science Co., Ltd
＜120〉people miR-149 antisense nucleic acid and application thereof
<130>DI09-1420-XC37
<160>8
<170>PatentIn version 3.3
<210>1
<211>89
<212>RNA
＜213〉people microRNA-149 precursor sequence
<400>1
gccggcgccc gagcucuggc uccgugucuu cacucccgug cuuguccgag gagggaggga 60
gggacggggg cugugcuggg gcagcugga 89
<210>2
<211>23
<212>RNA
<213>hsa-miR-149
<400>2
ucuggcuccg ugucuucacu ccc 23
<210>3
<211>21
<212>RNA
<213>hsa-miR-149*
<400>3
agggagggac gggggcugug c 21
<210>4
<211>23
<212>RNA
＜213〉artificial sequence
<220>
＜223〉antisense oligonucleotide
<400>4
gggagugaag acacggagcc aga 23
<210>5
<211>21
<212>RNA
＜213〉artificial sequence
<220>
＜223〉negative control antisense oligonucleotide
<400>5
caguacuuuu guguaguaca a 21
<210>6
<211>59
<212>DNA
＜213〉artificial sequence
<220>
＜223〉mir-149RT primer
<400>6
gtctgtatgg ttgttctgct ctctgtctca tccctatcta caaccataca gacgggagt 59
<210>7
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉mir-149FP primer
<400>7
ataactctgg ctccgtgtct tc 22
<210>8
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉mir-149RP primer
<400>8
tatggttgtt ctgctctctg tctc 24
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 '-UCUGGCUCCGUGUCUUCACUCCC-3 ' nucleotide sequence.
2. purposes as claimed in claim 1 is characterized in that, described antisense oligonucleotide sequence is 5 '-GGGAGUGAAGACACGGAGCCAGA-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 the 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 the cholesterol modification.
6. purposes as claimed in claim 5 is characterized in that, all Nucleotide 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.
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| WO2009036236A1 (en) * | 2007-09-14 | 2009-03-19 | The Ohio State University Research Foundation | Mirna expression in human peripheral blood microvesicles and uses thereof |
| CN101400361A (en) * | 2006-01-05 | 2009-04-01 | 俄亥俄州立大学研究基金会 | Microrna-based methods and compositions for the diagnosis, prognosis and treatment of lung cancer |
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| CN101091798A (en) * | 2007-04-17 | 2007-12-26 | 华东师范大学 | Application of meaning inversed miRNA-210 |
| WO2009036236A1 (en) * | 2007-09-14 | 2009-03-19 | The Ohio State University Research Foundation | Mirna expression in human peripheral blood microvesicles and uses thereof |
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