CN104415351A - Application of MicroRNA-429 in preparation of anti-hepatoma medicaments - Google Patents
Application of MicroRNA-429 in preparation of anti-hepatoma medicaments Download PDFInfo
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Abstract
The invention relates to application of MicroRNA-429 in preparation of anti-hepatoma medicaments. According to the application, the important role of miR-429 in the maintenance of the characteristics of hepatoma stem cells is firstly revealed; the multiplication, the self-renewal and the tumor formation capacity of the hepatoma stem cells of EpCAM+ can be enhanced by highly expressed miR-429; a reverse effect can be induced by virtue of the inhibition of the expression of miR-429. Therefore, miR-429 can serve as a new target spot for treating hepatoma, and a miR-429 inhibitor can serve as a hepatoma treatment medicament and is also capable of preventing the relapse and the transfer of the hepatoma.
Description
Technical field
The invention belongs to field of biological pharmacy, more specifically, the present invention relates to the Synthesis and applications of the medicines resistant to liver cancer that one is target spot with microRNA-429 (miR-429).
Background technology
Hepatocarcinoma is one of common malignant tumor, and mortality rate is high, and the patient that China dies from hepatocarcinoma every year accounts for 45% of whole world PLC mortality number.At present, the mechanism occurred about hepatocarcinoma is still unclear, and " stem cell " theory in recent years causes the concern of numerous scholar in the research of tumor.
Tumor stem cell hypothesis thinks to only have very little a part of cell to have unlimited multiplication capacity and differentiation potential, there is the ability causing tumor to occur, maintain tumor growth, keep Tumor Heterogeneity, such cell is tumor stem cell (cancer stem cell, CSC).It plays an important role in the generation of tumor, deterioration, transfer, recurrence.At present, researcher successively at Several Kinds of Malignancy, as qualification in the entity tumors such as leukemia, cerebral glioma, breast carcinoma, melanoma, cancer of pancreas, hepatocarcinoma, carcinoma of prostate, colon cancer, nasopharyngeal carcinoma is separated to the existence [1-5] of tumor stem cell.
Domestic and international experts and scholars are constantly probing into liver-cancer stem cell surface marker, and research shows that EpCAM is a labelling [6-8] of high oncogenicity tumor cell.For example, Kimura etc. [9] find that hepatocarcinoma EpCAM+ subgroup has stronger cloning efficiency.In body, induce tumor test finds in hyperimmune defect NOD/SCID/ γ cnull (NOG) Mus, and minimum 100 EpCAM+ cells can form tumor, and EpCAM-cell can not become tumor.
MiRNAs is a kind of endogenic, RNA of being about 22nt, and it causes its degraded or Translational repression by targeting mRNA and in animal and plant, plays important regulating action.MiRNAs is extensively present in primates, Rodents, birds, Fish, fly class, Vermes, plant, virus, more than 200 kind of statistics to the end of the year 2008 9000 kinds are had nearly from 2002, the discovery of miRNAs presents explosive growth, and constantly has the miRNAs in new species to be found.Mankind miRNAs gene only accounts for the 2-3% of gene number, but the protein expression of in human body 1/3 can be regulated and controled, this post-transcriptional control effect directly affects the function of these target genes, take part in physiology, the pathological process of the multiple systems comprising digestive system.The effect of miRNAs in digestive system relates in growth, aging, secretion, metabolism, viral infection, immunoreation, tumor etc., especially aobvious particularly important of the effect in tumor development as oncogene or antioncogene.
Research in the past shows, miR-200microRNA family member, especially miR-429 high expressed in kinds of tumors, and transforms play an important role [10-14] for maintenance epithelial phenotype, inducing cell substrate epithelium.
Indicated in prior art that the version of some antisensenucleic acidses is desirable, they also can play inhibitory action for corresponding target sequence.As Design and delivery of antisenseoligonucleotides to block microRNA function in cultured Drosophila and humancells (NATURE PROTOCOLS; VOL.3NO.10; 2008; Generally speaking 1537-1549) the multiple research of literature review, think, extend some bases be fine on the both sides of antisensenucleic acids.And between antisensenucleic acids and miRNA during affinity very high (as lock nucleic acid is modified), antisensenucleic acids can be punctured into about 2/3 length.
In the present invention, described " antisensenucleic acids " also comprises modified antisensenucleic acids, and described modification does not change the activity of antisensenucleic acids substantially, and more preferably, described modification can improve the activity of antisensenucleic acids, stability or therapeutic effect.Include but not limited to the modification of antisensenucleic acids: the modification that the modification of lock nucleic acid, methoxylation modification, peptide nucleic acid(PNA) modification, thio-modification, phosphate backbones are replaced by phospholipid connecting framework, these are modified is all that those skilled in the art are easy to realize.Preferably, the described lock nucleic acid that is modified to is modified.
Pharmaceutical composition
Present invention also offers a kind of compositions, it contains the miR-429 inhibitor of effective dose, and pharmaceutically acceptable carrier.Described compositions can be used for Hepatoma therapy, the propagation suppressing the liver-cancer stem cell of the EpCAM positive, self renewal and tumor Forming ability.Any aforesaid miR-429 or its inhibitor all can be used for the preparation of compositions.
As used herein, described " effective dose " refer to can to people and/or animal produce function or activity and can by people and/or animal the amount that accepts.Described " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent administration, comprises various excipient and diluent.This term refers to some medicament carriers like this: they itself are not necessary active component, and do not have undue toxicity after using.Suitable carrier is well known to those of ordinary skill in the art.Pharmaceutically acceptable carrier can contain liquid, as water, saline, buffer in the composition.In addition, in these carriers, also may there is complementary material, as filler, lubricant, fluidizer, wetting agent or emulsifying agent, pH buffer substance etc.Lipofectamine can also be contained in described carrier.
After the purposes obtaining miR-429 inhibitor described in cicada, can adopt multiple method well known in the art that described miR-429 inhibitor or the pharmaceutical composition containing it are delivered medicine to mammal.Include but not limited to: subcutaneous injection, intramuscular injection, percutaneous gives, local gives, implant, slow release gives.
The effective dose of miR-429 preparation of the present invention can change with order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be determined (such as passing through clinical trial) according to various factors by those of ordinary skill in the art.Described factor includes but not limited to: pharmacokinetic parameter such as bioavailability, metabolism, the half-life etc. of described miR-429 inhibitor; The order of severity of the disease that patient will treat, the body weight of patient, the immune state of patient, the approach etc. of administration.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.
Summary of the invention
The object of this invention is to provide the novel targets of liver cancer treatment.
Another object of the present invention is to provide MicroRNA-429 preparing the application in medicines resistant to liver cancer.
Another object of the present invention is to provide with miR-429 is the micromolecule nucleic acid drug of target spot and the application in liver cancer treatment thereof.
In a first aspect of the present invention, provide a kind of micromolecule nucleic acid drug being used for the treatment of hepatocarcinoma.Described medicine is the inhibitor of a class miR-429, by the material suppressing or stop miR-429 to be combined with its target gene, thus the propagation of the liver-cancer stem cell of the suppression EpCAM positive, self renewal and tumor Forming ability, play the effects such as the treatment to hepatocarcinoma, prevention of recurrence and transfer.
Described miR-429 inhibitor comprises (but being not limited to): the antisensenucleic acids of miR-429, the lock antisense nucleic acid of miR-429, siRNA; Or carry or express antisensenucleic acids, the lock antisense nucleic acid of miR-429, the construction (as expression vector) of siRNA of miR-429.
Described miR-429 inhibitor is: comprise SEQ ID NO:3 or the antisense nucleotide shown in SEQ ID NO:4, or its modified forms includes, but is not limited to: the modification that the modification of lock nucleic acid, methoxylation modification, peptide nucleic acid(PNA) modification, thio-modification, phosphate backbones are replaced by phospholipid connecting framework.
The present invention also provides the purposes of a kind of miR-429, for the target of the potential material as screening Hepatoma therapy; And a kind of method of screening the potential material of Hepatoma therapy; Preferably, described hepatocarcinoma is the hepatocarcinoma of hepatocarcinoma or EpCAM+.
The method of the potential material of described screening Hepatoma therapy, comprising:
(1) system of miR-429 is expressed with candidate substances process; With
(2) expression of miR-429 in described system is detected;
Wherein, if described candidate substances can reduce the expression of miR-429, then show that this candidate substances is the potential material of Hepatoma therapy.
In another preference, in described screening technique, step (1) comprising: in test group, candidate substances is joined in the system expressing miR-429; And/or
Step (2) comprising: the expression detecting miR-429 in the system of test group, and compares with matched group, and wherein said matched group is the system of the expression miR-429 not adding described candidate substances;
If in test group the expression of miR-429 statistically lower than (preferably remarkable lower than, as low by more than 20%, preferably low by more than 50%; Better is low by more than 80%) matched group, just show that this material standed for is the potential material of Hepatoma therapy.
In another preference, described candidate substances includes, but is not limited to: for disturbing molecule, nucleic acid inhibitor, binding molecule (as antibody or part), the micromolecular compound of miR-429 design.
In another preference, described system is selected from: cell system (as expressed the cell of miR-429) (or cell culture system), subcellular fraction system, solution system, organizational framework, organ systems or animal system.
In another preference, described method also comprises: carry out further cell experiment and/or animal experiment, to select further from candidate substances and to determine for the useful material of Hepatoma therapy to the potential material obtained.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, miR-429 promote the ratio of EpCAM+ cell.
Fig. 2, process LAN and interference miR-429 express the impact formed Spheroids.
Fig. 3, miR-429 inhibitor is to the inhibitory action of tumor growth.
Detailed description of the invention
The present inventor, through studying for a long period of time, discloses miR-429 first and play important effect in maintenance liver-cancer stem cell characteristic.High expressed miR-429 can strengthen the propagation of the liver-cancer stem cell of EpCAM+, self renewal and tumor Forming ability.Contrary, in the liver-cancer stem cell of EpCAM+, suppress the expression of miR-429 can cause contrary effect.Therefore, miR-429 can be used as the novel targets of liver cancer treatment, suppresses the micromolecule nucleic acid drug of miR-429 to can be used for the effective means of liver cancer patient postoperative adjuvant therapy, plays preventive effect simultaneously to liver cancer recurrence and transfer.
MiR-429 and uses thereof
In the present invention, described miR-429 is the micro ribonucleic acid (SEQID NO:1) with nucleotide sequence as follows:
miR-429:5’-UAAUACUGUCUGGUAAAACCGU-3’；
MiR-429 is one microRNA known in the art (miRNA) micromolecule, and it is useful for rna regulation.But before this area, the dependency between miR-429 and the stem cell properties of hepatocarcinoma is not studied.
MiR-429 can be separated from cell, or obtain by the mode of synthetic.After the sequence obtaining cicada miR-429 or its precursor, those skilled in the art can prepare miR-429 or its precursor easily.
In a particular embodiment of the present invention, utilize the method such as Cell Biology Experiment and animal pattern experiment in vivo, find with miR-429 be the micromolecule nucleic acid drug of target spot at the New function preparing the application aspect in antitumor drug, and it to be carried out the following studies:
1, gone out the liver-cancer stem cell of EpCAM+ by magnetic bead sorting, by miR-429 process LAN and disturb it, verify its impact for liver-cancer stem cell characteristic.
Following arbitrary sequence is at least comprised in a miR-429 process LAN reagent that () wherein uses:
SEQ ID NO:1：5’-UAAUACUGUCUGGUAAAACCGU-3’
SEQ ID NO:2：5’-TAATACTGTCTGGTAAAACCGT-3’
The process LAN reagent used is comprise the nucleic acid fragment of above-mentioned arbitrary sequence or the nucleic acid fragment through modifying;
Following arbitrary sequence is comprised in b miR-429 inhibitor that () wherein uses:
SEQ ID NO:3：5’-ACGGUUUUACCAGACAGUAUUA-3’
SEQ ID NO:4：5’-ACGGTTTTACCAGACAGTATTA-3’
The inhibitor used is comprise the nucleic acid fragment of above-mentioned arbitrary sequence or the nucleic acid fragment through modifying;
C the experiment of () process LAN shows that miR-429 can promote the self renewal of liver-cancer stem cell, propagation and chemoresistance; In the liver-cancer stem cell of EpCAM+, suppress the expression of miR-429 can cause contrary effect.
2, by the subcutaneous lotus tumor experiment of NOD-SCID mouse, in body, level verification suppresses the micromolecule nucleic acid drug of miR-429 (comprising the nucleic acid fragment of SEQ ID NO:3 or the arbitrary sequence of SEQ ID NO:4 or the nucleic acid fragment through the modifying) effect in liver cancer treatment.Research finds to suppress the micromolecule nucleic acid drug of miR-429 to have remarkable inhibitory action for the growth of hepatocarcinoma.
Above experimental result judges, miR-429 is the target spot that a hepatocarcinoma molecular therapy is new, suppresses the micromolecule nucleic acid drug of miR-429 to can be used for Hepatoma therapy, and prevention of postoperative recurrence and transfer.
Therefore, miR-429 be one new, with the closely-related drug target of stem cell properties of hepatocarcinoma.Various treatment meanss for miR-429 can as the novelty of Hepatoma therapy and effective means.
MiR-429 is the drug screening of target spot
After the Close relation of stem cell properties obtaining cicada miR-429 and hepatocarcinoma, can screen based on this feature the material suppressing miR-429.Afterwards, can find from described material for the really useful medicine of Hepatoma therapy.
Therefore, the invention provides a kind of method of screening the potential material of Hepatoma therapy, described method comprises: the system expressing miR-429 with candidate substances process; With the expression or the activity that detect miR-429 in described system; If described candidate substances can suppress expression or the activity of miR-429, then show that this candidate substances is the potential material of Hepatoma therapy.The system of described expression miR-429 can be such as cell (or cell culture) system, and described cell can be the cell of endogenous expression miR-429; It can be maybe the cell of recombinant expressed miR-429.The system of described expression miR-429 can also be subcellular fraction system, solution system, organizational framework, organ systems or animal system (as animal model, the animal model of preferred non-human mammal, as Mus, rabbit, sheep, monkey etc.) etc.
In optimal way of the present invention, when screening, in order to be easier to observe the expression of miR-429 or the change of activity, also can arrange matched group, described matched group can be the system of the expression miR-429 not adding described candidate substances.
As optimal way of the present invention, described method also comprises: carry out further cell experiment and/or animal experiment, to select further and to determine for the really useful material of Hepatoma therapy to the potential material obtained.
On the other hand, present invention also offers the potential material of the Hepatoma therapy adopting described screening technique to obtain.The material that these Preliminary screening go out can form a screening storehouse so that people finally can therefrom filter out can for suppressing the expression of miR-429 and activity, and then the material that Hepatoma therapy is useful.
MiR-429 regulator and uses thereof
Based on the above-mentioned new discovery of the present inventor, the invention provides a kind of purposes of miR-429 inhibitor, for the preparation of the compositions of Hepatoma therapy.Described miR-429 inhibitor also for: suppress the propagation of the liver-cancer stem cell of the EpCAM positive, self renewal and tumor Forming ability.
As used herein, described " miR-429 inhibitor " includes nucleic acid inhibitor, antagonist, lower adjustment, blocker, blocker etc., as long as they can lower the expression of miR-429 or its precursor.They can be compound, chemical small molecule, biomolecule.Described biomolecule can be nucleic acid level (comprising DNA, RNA), also can be protein level.
Described miR-429 inhibitor refers to that any miR-429 of prevention (particularly wherein in conjunction with critical sites) or its precursor are combined with its targeting sequence, reduce the activity of miR-429 or its precursor, reduce the stability of miR-429 or its precursor, lower the expression of miR-429 or its precursor, reduce the material of miR-429 or its precursor effective acting time, these materials all can be used for the present invention, as for lowering the useful material of miR-429, thus can be used for Hepatoma therapy, suppress the propagation of the liver-cancer stem cell of the EpCAM positive, self renewal and tumor Forming ability.Such as, described inhibitor is: nucleic acid inhibitor, protein inhibitor, antibody, part, nuclease, nucleic acid binding molecule, as long as it can lower the expression of miR-429.
As a kind of optimal way of the present invention, described inhibitor is nucleic acid inhibitor.Preferably, described nucleic acid inhibitor is combined with its targeting sequence by stoping miR-429, thus plays a role.The antisensenucleic acids of described nucleic acid inhibitor is such as selected from (but being not limited to): miR-429, the lock antisense nucleic acid of miR-429; Peptide nucleic acid(PNA); SiRNA (reticent miR-429 precursor).
As optimal way of the present invention, the inhibitor of described miR-429 is the antisensenucleic acids of miR-429.As used herein, " antisensenucleic acids " is also called " antisensenucleic acids " or " antisense oligonucleotide (AS-ONs; antisense-oligonucleotides) " or " antisense drug ", refer to that length is about the DNA molecular of 15-30 base, their modified form of RNA molecule or their analog, can be complementary with mRNA.
Those skilled in the art all understand, and according to antisensenucleic acids provided by the invention, can carry out suitable change and retain its activity, these versions all can be used for the present invention.Any antisensenucleic acids playing the effect of suppression or reticent miR-429 is all used in the present invention, and its type is not limited to DNA or RNA.Such as, the antisensenucleic acids of described miR-429 is and the sequence of the miR-429 sequence that (being preferably complete) is complementary substantially.Such as, the antisensenucleic acids of described miR-429 and SEQ ID NO:3 or the sequence shown in SEQ ID NO:4 have the homogeny of more than 80%, preferably there is the homogeny of more than 85%, more preferably there is the homogeny of more than 90%, there is the homogeny of more than 95% best, as having the homogeny of more than 96%, 97%, 98% or 99%; Or the antisensenucleic acids of described miR-429 is the fragment of sequence shown in SEQ ID NO:3 or SEQID NO:4; They all have the identical function of the antisensenucleic acids of sequence shown in SEQ ID NO:3 or SEQ ID NO:4.
The preparation of embodiment 1, miR-429 process LAN fragment and suppression fragment
1, miR-429 process LAN reagent
According to miR-429 sequence, design miR-429 process LAN reagent, is selected from following arbitrary sequence:
SEQ ID NO:1：5’-UAAUACUGUCUGGUAAAACCGU-3’；
SEQ ID NO:2：5’-TAATACTGTCTGGTAAAACCGT-3’；
The miR-429 inhibitor wherein used, is selected from following arbitrary sequence:
SEQ ID NO:3：5’-ACGGUUUUACCAGACAGUAUUA-3’；
SEQ ID NO:4：5’-ACGGTTTTACCAGACAGTATTA-3’。
Embodiment 2, miR-429 process LAN and disturb it, verify its impact for liver-cancer stem cell characteristic
EpCAM is the surface marker of liver-cancer stem cell.With U.S. sky Ni (Miltenyi Biotech) magnetic bead in conjunction with EpCAM antibody, from Bel7402 HCCLM3, sorting obtains EpCAM+ liver-cancer stem cell, subsequently external source proceed to miR-429 process LAN fragment or suppress fragment, specific experiment method and result as follows:
Appropriate HCCLM3 (EpCAM+) cell (500 μ lDMEM+10% (v/v) FBS) is inoculated in 24 hole culture dishs, converge rate after making it to cultivate 24h and reach 60-70%, 50 μ l normal saline are added respectively in different 1.5ml centrifuge tube, add 1 μ g miR-429 process LAN nucleic acid fragment or suppression fragment and 2 μ l JetPEI respectively and mix, DNA solution is added with by PEI solution, vibration mixing, room temperature places 15-20min, plasmid DNA-liposome complex is dropped to cell surface, puts 5%CO
2, in 37 DEG C of incubators, cultivate 24-48h.
100nM miR-429 process LAN fragment is entered after 4 days at HCCLM3 (EpCAM+) transit cell, analyze by flow cytometer Annexin V method, result, the ratio obtaining EpCAM+ cell rises to 60.8% from 3.86%, as Fig. 1 (application process LAN nucleic acid fragment SEQ ID NO:2).This result illustrates that miR-429 promotes the formation rate of the EpCAM+ subgroup of hepatoma carcinoma cell, promotes that tumor is formed.
Next, after observing process LAN and interference miR-429 expression, the liver-cancer stem cell HCCLM3 (EpCAM+) sub-elected is formed to the impact of Spheroids.Tumor many cells ball (Multicellular TumorSpheroids) is a kind of external three-dimensional nodule cell culture model of classics, by simulation three-dimensional cell network, cell and substrate, interaction between cell and cell, thus corresponding pathophysiology characteristic in energy simulating human tumor tissues, according to its growth and morphology dynamics, it is similar to tumor and occurs early stage without the region away from blood vessel in hemangioma or solid tumor mass).3000 liver-cancer stem cells after proceeding to miR-429 process LAN fragment or suppression fragment are inoculated on low adhesion culture plate, cultivate and within 10 days, find afterwards compared with respective contrast, Spehroids quantity and miR-429 are expressed as direct ratio, as Fig. 2 (apply process LAN nucleic acid fragment SEQ ID NO:2 respectively and suppress fragment SEQ ID NO:4).This result illustrates, suppressing miR-429 to express can significantly reduce Spheroids quantity, thus can Tumor suppression tissue growth.
Embodiment 4, miR-429 suppress the application of fragment in liver cancer animal model treatment
In order to level in vivo verify further miR-429 inhibitor liver EpCAM+T-ICs tumor is formed after therapeutic effect, the present inventor has prepared NOD-SCID mice subcutaneous lotus tumor model.
The preparation method of the HCCLM3 cell line of stably express luciferase is as follows:
(1) using JetPEI(purchased from French PolyPlus company, article No. 101-10N) pGL3-control vector plasmid (purchased from American Pu Luomaige company, article No. is E1741) is transfected in HCCLM3 hepatoma carcinoma cell by transfection reagent;
(2) after transfection 48 hours, use antibiotic G418(purchased from American Life Technology company, concentration was 800ug/ml) screen;
(3) the HCCLM3 hepatoma cell line (HCCLM3-Luc) obtaining stably express luciferase after 3 weeks is screened, for follow-up test.
NOD-SCID mice subcutaneous lotus tumor method for establishing model is as follows:
(1) magnetic bead sorting goes out HCCLM3-Luc (EpCAM+) cell, plants in 6 orifice plates;
(2) HCCLM3-Luc (EpCAM+) cell uses the miR-429 inhibitor process one day of 40 μMs;
(3) positive controls is that HCCLM3-Luc (EpCAM+) cell uses normal saline process one day;
(4) trypsin digestion cell of 0.25-0.5%, 900rpm, 3min, PBS re-suspended cell;
(5) cell counting, adjustment volume, makes in every 100 μ l PBS containing 2 × 10
4individual cell;
(6) will containing 2 × 10
4the miR-429 inhibitor processed group of HCCLM3-Luc (EpCAM+) cell and normal saline processed group to plant NOD-SCID mice (purchased from The 2nd Army Medical College animal center) right side of trunk respectively subcutaneous;
(7) a situation arises to record tumor, and tumor cell plantation carries out living imaging after within 6 weeks, becoming tumor, and disconnected neck puts to death animal, collects tumor sample and plasma sample.
Living imaging analytical method is as follows:
(1) tumor-bearing mice anesthesia latter 5 minutes use balances are weighed in.According to the standard of per kilogram of body weight 15mg luciferase substrate (Beetle Luciferin, Potassium Salt, purchased from American Pu Luomaige company, article No. is E1605), the luciferase substrate solution of the 30mg/ml concentration of injection respective volume.
(2) after injected fluorescein enzyme substrate solution, mice is placed in small animal living body imager (PE company) and imaging is carried out to anesthetized mice.
(3) chemiluminescence detection parameter is: imaging time 10 minutes, and bin value is 4*4.
(4) visible detection parameter is: imaging time 30 milliseconds, and bin value is 4*4.
(5) living imaging instrument embedded software is adopted to analyze the intensity of chemiluminescence signal and area after imaging, the chemiluminescence signal value of more different group mice.
Found that, miR-429 suppresses fragment to have remarkable inhibitory action for the growth of tumor, as Fig. 3 (application suppresses fragment SEQ ID NO:4).
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
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Claims (10)
- The purposes of the inhibitor of 1.miR-429, for the preparation of the medicine of Hepatoma therapy.
- 2. purposes as claimed in claim 1, is characterized in that, described medicine also for: suppress the propagation of the liver-cancer stem cell of the EpCAM positive, self renewal and tumor Forming ability.
- 3. purposes as claimed in claim 1, is characterized in that, miR-429 inhibitor is the material suppressing or stop miR-429 to be combined with its target gene.
- 4. purposes as claimed in claim 1, it is characterized in that, described miR-429 inhibitor comprises: the antisensenucleic acids of miR-429, the lock antisense nucleic acid of miR-429, siRNA; OrCarry or express the antisensenucleic acids of miR-429, the lock antisense nucleic acid of miR-429, the construction of siRNA.
- 5. purposes as claimed in claim 4, it is characterized in that, described miR-429 inhibitor comprises: the antisense nucleotide comprising sequence shown in SEQ ID NO:3 or SEQ ID NO:4, or its modified forms.
- 6. purposes as claimed in claim 1, it is characterized in that, described hepatocarcinoma is the hepatocarcinoma of hepatocarcinoma or the EpCAM positive.
- 7. a miR-429 inhibitor, is characterized in that, it is: the antisense nucleotide comprising sequence shown in SEQ ID NO:3 or SEQ IDNO:4, or its modified forms.
- 8. a purposes of miR-429, for the target of the potential material as screening Hepatoma therapy; Preferably, described hepatocarcinoma is the hepatocarcinoma of hepatocarcinoma or EpCAM+.
- 9. screen a method for the potential material of Hepatoma therapy, described method comprises:(1) system of miR-429 is expressed with candidate substances process; With(2) expression of miR-429 in described system is detected;Wherein, if described candidate substances can reduce the expression of miR-429, then show that this candidate substances is the potential material of Hepatoma therapy.
- 10. method as claimed in claim 9, it is characterized in that, step (1) comprising: in test group, candidate substances is joined in the system expressing miR-429; And/orStep (2) comprising: the expression detecting miR-429 in the system of test group, and compares with matched group, and wherein said matched group is the system of the expression miR-429 not adding described candidate substances;If the expression of miR-429 is statistically lower than matched group in test group, just show that this material standed for is the potential material of Hepatoma therapy.
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| CN106997423A (en) * | 2016-07-22 | 2017-08-01 | 中国药科大学 | A kind of structure of medicine penetration PK model based on three-dimensional cell model and its application in drug evaluation |
| CN109646449A (en) * | 2018-10-17 | 2019-04-19 | 河南师范大学 | A kind of miR-429 instrumentality of liver cell and its application adjusted in liver cell drug is treated in preparation |
| CN114366814A (en) * | 2022-01-24 | 2022-04-19 | 南通大学 | Inhibitor of miR-27a-3p and application thereof |
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