CN102041256B - Human miR-486-5p antisense nucleic acid and application thereof - Google Patents

Human miR-486-5p antisense nucleic acid and application thereof Download PDF

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CN102041256B
CN102041256B CN 200910197380 CN200910197380A CN102041256B CN 102041256 B CN102041256 B CN 102041256B CN 200910197380 CN200910197380 CN 200910197380 CN 200910197380 A CN200910197380 A CN 200910197380A CN 102041256 B CN102041256 B CN 102041256B
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丁侃
张佩琢
东楠
李捷
沈孝坤
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Suzhou Genepharma Co ltd
Shanghai Institute of Materia Medica of CAS
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Shanghai Institute of Materia Medica of CAS
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Abstract

The invention discloses antisense oligonucleotide for suppressing expression of micro-ribonucleic acid (RNA)-486-5p and application thereof. The antisense oligonucleotide comprises a sequence of 5'-CUCGGGGCAGCUCAGUACAGGA-3', wherein the sequence can be specifically combined with human miR-486-5p. The antisense oligonucleotide can be ribonucleotide or deoxyribonucleotide and can modify any nucleotide. The miR-486-5p antisense oligonucleotide with the effect of suppressing growth of tumor cells can effectively suppress the expression of the miR-486-5p in human brain glioma cells U87 and simultaneously suppress the growth and proliferation of the cells, so that brain gliomas and other tumors with high miR-486-5p expression can be effectively treated.

Description

People miR-486-5p antisense nucleic acid and application thereof
Technical field
The present invention relates to biomedicine field.Particularly, the present invention relates to the purposes of a kind of small molecules microRNA (miRNA), relate in particular to the application of the antisense oligonucleotide of this miRNA.This antisense oligonucleotide can be complementary with people miR-150, thereby suppress the expression of people miR-150.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 20-22bp, 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 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, 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 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, also can play 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, the precursor molecule of its hairpin structure is similar with content in the healthy tissues in tumour, and this shows, 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, miR-21 expresses increase in glioblastoma multiforme.Expression amount is than the high 5-100 of healthy tissues doubly in tumor tissues for this gene.
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; MetastasisReviews 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.
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-486-5p, be used for efficient, low toxicity or suppress innocuously the expression of miR-486-5p, and then the treatment disease relevant with the miR-486-5p overexpression, comprise various noumenal tumours, various leukemia etc.
Another problem that the present invention will solve just provides above-mentioned antisense nucleic acid and suppress the application that miR-486-5p expresses and inhibition tumor cell is grown and bred in tumour cell.
The inventor has designed and synthesized a series of specificitys for the antisense nucleic acid of miR-486-5p 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 has designed a kind of antisense nucleic acid molecule that can be incorporated into miR-486-5p, in culturing cell U87, and the antisense nucleic acid cell growth ability that checking suppresses the miR-486-5p expression specificity, the impact 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 of inhibition tumor cell energy for growth, multiplication capacity, wherein the tumour cell of preferred miR-486-5p 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-486-5p, and described antisense oligonucleotide suppresses the expression of miR-486-5p in people's cell.Usually, continuous 13 ~ 22 nucleotide sequence complementations in the described antisense oligonucleotide and 5 ' UCCUGUACUGAGCUGCCCCGAG-3 '.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 is 5 ' CUCGGGGCAGCUCAGUACAGGA 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 the DNA cost of the synthetic 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-486-5p 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-486-5p expresses that suppresses.In the antisense nucleic acid research, various chemical modification methods are a lot.The antisense nucleic acid that the present invention adopts sulfo-, methoxy to modify, mainly be that the preclinical studies such as these the two kinds pharmacology of modifying the antisense nucleic acid of modes, 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, thus make antisense nucleic acid lose should by 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.
Above-mentioned antisense nucleic acid of the present invention has the effect that suppresses the miR-486-5p expression.After among the cell strain U87 that above-mentioned antisense nucleic acid is transfected into the miR-486-5p high expression level, 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.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with physiological saline or the aqueous solution that contains glucose and other assistant agents.
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:
(A) treatment human tumor growth;
(B) the treatment human body is crossed with miR-486-5p and is expressed relevant disease.
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 (RNase H), 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 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 ~ 22bp.
Description of drawings
Fig. 1 has shown the expression of miR-486-5p in the miR-486-5p antisense oligonucleotide inhibition tumor cell U87 cell, A is the expression of miR-486-5p behind the transfection miR-486-5p antisense oligonucleotide, and B is the expression of miR-486-5p behind the transfection negative control antisense oligonucleotide; C is the expression of reference gene U6 behind the transfection miR-486-5p antisense oligonucleotide, and D is the expression of reference gene U6 behind the transfection negative control antisense oligonucleotide; E is miR-486-5p inhibition histogram behind the transfection antisense oligonucleotide, and the miR-486-5p expression behind the sample 6 expression transfection miR-486-5p antisense oligonucleotides wherein, NC represent miR-486-5p expression behind the transfection negative control.
Embodiment
Antisense oligonucleotide of the present invention, continuous 13 ~ 22 nucleotide sequence complementations in its sequence and 5 ' UCCUGUACUGAGCUGCCCCGAG-3 ', and should be not complementary with the RNA sequence of other genes.In a preferred embodiment of the invention, the sequence of described antisense oligonucleotide is 5 ' CUCGGGGCAGCUCAGUACAGGA 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 as and 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-486-5p is reached 90%, to the inhibiting rate of growth of tumour cell near 50%.
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.
Embodiment 1, miR-486-5p antisense nucleic acid suppress the expression of miR486-5p
Real-time quantitative fluorescence detects test and is finished by Shanghai JiMa pharmacy Technology Co., Ltd, and concrete experimental procedure comprises:
Cell cultures: the U87 cell, the 10%FBS-DMEM culture medium culturing, 37 ℃, 5%CO2 cultivates.
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. the Opti-MEM that does not contain serum with 50 μ 1
Figure G2009101973801D00061
The I substratum dilutes respectively the negative control of miR-486-5p inhibitor, negative control, FAM mark, and final concentration is 50nM, mixing gently, and 3 multiple holes are established in each transfection;
B. mixing Lipofecta mine gently before using TM2000, then get the Opti-MEM that 2 μ l are diluted to 50 μ l
Figure G2009101973801D00062
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 72h,
Total RNA extracts:
1) centrifugal collecting cell adds 500 μ l Ezol 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, 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 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% washing with alcohol 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-486-5p respectively, preparation cDNA template.Damping fluid and enzyme used in the reverse transcription are Promega company product.
(i). the reverse transcription system:
Reagent name Consumption/pipe
5×Reverse Transcription buffer 4μl
RT Primer Mix(U6,hsa-mir-486-5p) (1μM) 1.25μl
dNTP(10mM) 0.75μl
RNA 2μl
RTase(200/μl) 0.5μl
DEPC H
20 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 RNase/DNase free ddH 2O, mixing.
(2). the quantitative fluorescent PCR system:
Reagent name Consumption/pipe
2×PCR Master Mix 10μl
F Primer(20μM) 0.2μl
R Primer(20μM) 0.2μl
Template 2μl
rTaq DNA polymerase(5U/ul) 0.2μl
ddH 2O 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.
Embodiment 2, MiRNA antisense oligonucleotide are that U87 suppresses active detection to the neuroglia cell of human oncocyte
Cell cultures:
The U87 cell, the 10%FBS-DMEM culture medium culturing, 37 ℃, 5%CO2 cultivates.Collect the good U87 cell of growth conditions, centrifugal counting is with 2 * 10 3Every hole is laid in 96 orifice plates, and 37 ℃, 5%CO2 cultivates.
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. the Opti-MEM that does not contain serum with 25 μ l The I substratum dilutes respectively the negative control of miR-486-5p antisense oligonucleotide, negative control, FAM mark, and final concentration is 50nM, mixing gently, and 3 multiple holes are established in each transfection;
B. mixing Lipofecta mine gently before using TM2000, then get the Opti-MEM that 1 μ l is diluted to 25 μ l
Figure G2009101973801D00082
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;
4) 37 ℃, 5%CO 2The incubator overnight incubation is changed the substratum that contains 10% foetal calf serum and is continued to cultivate 72h, continues to cultivate 72 hours.
The MTT experiment:
Add MTT (Sigma) 5mg/ml (physiological saline with 0.9% is prepared) for preparing in the cell that obtains in the previous step, every hole adds 20 μ l hatches after 4 hours and sucks substratum and MTT, and every hole adds DMSO100 μ l and reads the absorbance of 0D570-0D630 by microplate reader.
The sample negative control
0.43 0.84
0.37 0.64
0.44 0.71
Calculate inhibiting rate:
ln hibitionrate ( % ) = NC ( OD 570 - OD 630 ) - MicroRNAinhibitor ( OD 570 - OD 630 ) NC ( OD 570 - OD 630 ) × 100 %
Calculating inhibiting rate is 43.45 ± 5.06%.

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 '-UCCUGUACUGAUCUGCCCCGAG-3 ' nucleotide sequence.
2. purposes as claimed in claim 1 is characterized in that, described antisense oligonucleotide sequence is 5 ' CUCGGGGCAGCUCAGUACAGGA-3 '.
3. claim 1 or 2 described purposes is characterized in that, described antisense oligonucleotide is the modification type of antisense oligonucleotide.
4. purposes as described 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, the cholesterol modification.
6. purposes as claimed in claim 5 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.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009105759A2 (en) * 2008-02-21 2009-08-27 The Board Of Regents Of The University Of Texas System Micro-rnas that modulate smooth muscle proliferation and differentiation and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009105759A2 (en) * 2008-02-21 2009-08-27 The Board Of Regents Of The University Of Texas System Micro-rnas that modulate smooth muscle proliferation and differentiation and uses thereof

Non-Patent Citations (2)

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Hilah Gal et al..MIR-451 and Imatinib mesylate inhibit tumor growth of Glioblastoma stem cells.《Biochemical and Biophysical Research Communications》.2008,第376卷(第1期),86-90. *
杨鹏远.反义寡核苷酸类药物的研究进展.《国外医学药学分册》.2002,第29卷(第4期),193-197. *

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