CN108251422A - Pass through the method and drug of miR-6069 progress anticancers and its application - Google Patents
Pass through the method and drug of miR-6069 progress anticancers and its application Download PDFInfo
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- CN108251422A CN108251422A CN201611245389.1A CN201611245389A CN108251422A CN 108251422 A CN108251422 A CN 108251422A CN 201611245389 A CN201611245389 A CN 201611245389A CN 108251422 A CN108251422 A CN 108251422A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
Abstract
The present invention relates to biomedicine fields, disclose through the method and drug of the progress anticancers of miR 6069 and its application.Specifically, the invention discloses the application of the function of inhibiting miR 6069 and/or the expression of the target gene of promotion miR 6069 in prevention and/or treating cancer and the disease similar to its symptom, particularly preparing for the application in prevention and/or the drug and/or food for the treatment of cancer and the disease similar to its symptom.The inhibitor of miR 6069 provided by the invention can fully inhibit the function of miR 6069 and promote the expression of its target gene.This explanation can effectively prevent when by the inhibitor for individual administration and/or treat that miR 6069 is high to express and/or its target gene crosses disease caused by low expression.
Description
Technical field
The present invention relates to biomedicine fields, and in particular, to inhibits the function of miR-6069 and/or promotes miR-6069
Target gene application of the expression in prevention and/or treating cancer and the disease similar to its symptom, a kind of miR-6069 suppressions
Preparation, a kind of method of the expression of function of inhibiting miR-6069 and/or the target gene for promoting miR-6069, a kind of pharmaceutical composition
Object, it is a kind of prevent and/or the method for the treatment of cancer and they inhibiting the function of miR-6069 and/or promoting miR-6069
Application in the expression of target gene and they in the function of preparing for inhibiting miR-6069 and/or promote miR-6069's
Application in the drug of the expression of target gene.
Background technology
MicroRNA (microRNA, miRNA) is the non-coding RNA molecule that a kind of length is 16-25nt
(www.mirbase.org), can by with target gene partial complementarity match identify and silencing of target genes rna expression and/or
Protein expression.After ripe microRNA is loaded on the silencing complex (RISC) of RNA inductions, by base pairing come with target base
Because the complementary series in mRNA 3'-UTR is combined, so as to cause the degradation of mRNA and/or inhibit the translation of its protein.It is micro-
The nucleotide of the second at RNA 5' ends to the 8th is referred to as " core sequence ", this seven nucleotide and the mutual of target gene recruit
To being the key that identification target gene, pairing degree is higher, bigger with reference to the possibility with adjusting target gene and ability.It is micro- simultaneously
The complementary pairing of other sequences and target gene except RNA " core sequence " can also enhance its energy for combining and regulating and controlling target gene
Power.It is the expression for identifying by non-fully matching and adjusting target gene just because of microRNA, a microRNA is just enabled to exist
One adjusts multiple target genes to some extent simultaneously into the cell.
More and more document report microRNAs cancer generation, shift important function, and inhibition or overexpression
Certain microRNAs can play the role of anticancer.Therefore, it is quite necessary to develop the anticancer drug based on microRNA.
Invention content
The purpose of the invention is to develop the new anticancer drug based on microRNA.
To achieve these goals, in a first aspect, the present invention provides the function of inhibiting miR-6069 and/or promoting miR-
Application of the expression of 6069 target gene in prevention and/or treating cancer and the disease similar to its symptom.
Second aspect, the present invention also provides a kind of miR-6069 inhibitor, wherein, the miR-6069 inhibitor is
The nucleotide of the promoter activity of the antisense oligonucleotides of miR-6069, the siRNA of miR-6069 and inhibition miR-6069
And the like.
The third aspect, the present invention also provides a kind of function of inhibiting miR-6069 and/or the target bases for promoting miR-6069
The method of the expression of cause, this method include：MiR-6069 inhibitor is contacted with expressing the target cell of miR-6069, wherein, institute
MiR-6069 inhibitor is stated as above-mentioned miR-6069 inhibitor.
Fourth aspect, the present invention also provides a kind of pharmaceutical composition, wherein, which contains above-mentioned miR-
6069 inhibitor and pharmaceutically acceptable carrier.
5th aspect, the present invention also provides a kind of prevention and/or the method for the treatment of cancer, wherein, this method includes：Suppression
The expression of the function of miR-6069 processed and/or the target gene of promotion miR-6069；Or by above-mentioned miR-6069 inhibitor and/or
Aforementioned pharmaceutical compositions are administered to subject.
6th aspect, the present invention also provides above-mentioned miR-6069 inhibitor, above-mentioned inhibition miR-6069 function and/or
The method of the method for expression of the target gene of miR-6069, aforementioned pharmaceutical compositions, above-mentioned prevention and/or treating cancer is promoted to exist
Application in the expression of the function of inhibiting miR-6069 and/or the target gene for promoting miR-6069.
7th aspect, the present invention also provides above-mentioned miR-6069 inhibitor, aforementioned pharmaceutical compositions to prepare for pressing down
Application in the drug of the expression of the function of miR-6069 processed and/or the target gene of promotion miR-6069.
The present invention, can be abundant by the way that miR-6069 inhibitor is contacted with the target cell of excessively high expression miR-6069
Inhibit miR-6069 function (including inhibiting the combination of miR-6069 and its target gene or the expression quantity of reduction miR-6069,
So as to inhibit the function of miR-6069) and/or promote its target gene (PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4,
SASH1, FBXO31, CIDEB, TMEM127, SKI and PLEKHO1) at least two expression.This explanation is worked as the inhibitor
When being administered for individual, can effectively prevent and/or treat miR-6069 it is excessively high express and/or its target gene (PPM1F,
TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1, FBXO31, CIDEB, TMEM127, SKI and PLEKHO1) in extremely
Few two kinds of diseases crossed caused by low expression, for example, various cancers or the disease similar to its symptom, particularly liver cancer, lung cancer
At least one of with cutaneum carcinoma.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Attached drawing is to be used to provide further understanding of the present invention, and a part for constitution instruction, with following tool
Body embodiment is used to explain the present invention, but be not construed as limiting the invention together.In the accompanying drawings：
The antisense oligonucleotides that miR-6069 is shown in Fig. 1 inhibits/kills the result figure of liver cancer cells HuH-7；
The antisense oligonucleotides that miR-6069 is shown in Fig. 2 inhibits/kills the result figure of hepatocellular carcinoma H22；
The antisense oligonucleotides that miR-6069 is shown in Fig. 3 inhibits/kills the result figure of lung cell A549；
The result that the antisense oligonucleotides of miR-6069 influences normal human fibroblastic growth is shown in Fig. 4
Figure；
Result figures of the miR-6069 to the inhibiting effect of expression of target gene is shown in Fig. 5；
The antisense oligonucleotides of the antisense oligonucleotides of miR-6069 and the miR-6069 of mispairing is shown to miR- in Fig. 6
The result figure of the inhibiting effect of 6069 functions；
The antisense oligonucleotides that the miR-6069 of thio-modification is shown in Fig. 7 inhibits/kills the knot of liver cancer cells HuH-7
Fruit is schemed.
Specific embodiment
The specific embodiment of the present invention is described in detail below.It is it should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood to comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It between the endpoint value of a range and individual point value and can be individually combined with each other between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
In a first aspect, the present invention provides the function of inhibiting miR-6069 and/or the tables for the target gene for promoting miR-6069
Up to the application in prevention and/or treating cancer and the disease similar to its symptom, particularly preparing for preventing and/or control
Treat the application in the drug and/or food of cancer and the disease similar to its symptom.
In the present invention, the term function of miR-6069 " inhibit " refer to inhibit miR-6069 and its target gene combination or
Person reduces the expression quantity of miR-6069.
In the present invention, the cancer can be at least one of liver cancer, lung cancer and cutaneum carcinoma.
In situations where it is preferred, miR-6069 has SEQ ID NO：Nucleotide sequence shown in 1.
In the present invention, the target gene of the miR-6069 can be PPM1F, TNFSF15, BCAP29, HRK, BID,
In GRAMD4, SASH1, FBXO31, CIDEB, TMEM127, SKI and PLEKHO1 at least two, at least three kinds, at least four,
At least five kinds, at least six kinds, at least seven kinds, at least eight kinds, at least nine kinds, at least ten kinds, at least ten one kind or 12 kinds.
Second aspect, the present invention also provides miR-6069 inhibitor, which can inhibit miR-
The expression of 6069 function and/or the target gene of promotion miR-6069 includes but not limited to following several：
1) micromolecular compound
MiR-6069 inhibitor includes but is not limited to naturally occurring or artificial synthesized micromolecular compound, this kind of small point
Sub- compound directly acts on miR-6069 and to be increased by the expression quantity of the miR-6069 target genes adjusted, typically molecule
Amount is more than 50 and less than the organic compound of 2500 dalton.This kind of candidate compound possesses and protein, particularly hydrogen bond phase
The functional group of interaction, and generally comprise at least one amine, carbonyl, hydroxyl or carboxylic group.These small molecules miR-6069
Inhibitor can be found by suitable screening technique or other methods.
2) antisense oligonucleotides
The antisense oligonucleotides can by with miR-6069 directly in conjunction with inhibiting the function of miR-6069, and then promote
Into the expression of the target gene of miR-6069, the antisense oligonucleotides includes antisense RNA and antisense DNA.Preferably, the antisense
Oligonucleotides is complementary with miR-6069, has the length of 12-30 nucleotide, and has what is at least held with the 5 ' of miR-6069
The sequence of 1-8 nucleotide complementations.
It is well known in the art that microRNA can identify the table of simultaneously silencing of target genes by being matched with target gene partial complementarity
It reaches and/or translates, similarly, miR-6069 also can be by combining the nucleotide sequence with its partial complementarity and Reverse transcriptase
The function of its own, so as to raise the expression of the target gene of miR--6069.Therefore, in the present invention, term " complementation " not only includes
Complete complementary further includes partial complementarity (or non-fully complementary).In addition, 60% complementation refers to using the sequence of miR-6069 as base
Standard, the base complementrity with therein 60%.
Therefore, the antisense oligonucleotides has following nucleotide sequence：
a)SEQ ID NO：Nucleotide sequence shown in 2；
B) in SEQ ID NO：In nucleotide sequence shown in 2 through lack, replace or add one or several nucleotide and
With miR-6069 complete complementaries or the nucleotide sequence of partial complementarity.
When in the case of non-fully complementary, that is, when the antisense oligonucleotides is by SEQ ID NO：Shown in 2
Nucleotide sequence in the case of the nucleotide sequence that is obtained through lacking, replacing or adding one or several nucleotide, mutual
Mend nucleotide region in, the antisense oligonucleotides preferably should with miR-6069 at least have 60%, 65%, 70%, 75%,
80%th, 85%, 90% or 95% complementation.More preferably, in the nucleotide region of the 1-8 positions at the 5 ' ends of miR-6069,
The antisense oligonucleotides at most has the mispairing of 2 nucleotide.
As described above, in the case where the antisense oligonucleotides and miR-6069 are non-fully complementary, further preferably
, with SEQ ID NO：2 compare, and will at most have 10,9,8,7,6,5,3,2 or 1 cores in length
The difference of thuja acid.
In a preferred case, non-fully complementary antisense oligonucleotides has SEQ ID NO with miR-6069：3
The nucleotide sequence.
It is in addition, few to improve the antisense the invention also includes some conventional modifications are carried out to the antisense oligonucleotides
The stability and activity of nucleotide, these are all belonged to the scope of the present invention.
In the present invention, the antisense oligonucleotides contains nucleotide groups (or nucleotide residue) as basic structure list
Member, the nucleotide groups contain phosphate group, ribose groups and base, and under preferable case, the antisense oligonucleotides contains
The nucleotide groups of at least one modification.The nucleotide groups of the modification will not cause the antisense oligonucleotides to inhibit miR-
The function of the expression of 6069 function and/or the target gene of promotion miR-6069 is lost.
In the present invention, the nucleotide groups of the modification are the nucleotide that phosphate group and/or ribose groups are modified
Group.For example, the modification of phosphate group refers to modify the oxygen in phosphate group, including thio-modification, boranated modification
Deng as respectively with the oxygen in sulphur and borine displacement phosphate group.Phosphate group is modified the structure for being capable of stabilization of nucleic acids, keeps alkali
The high specific and high-affinity of basigamy pair.Preferably, the nucleotide groups of thio-modification refer to wherein all di-phosphate esters
Nonbridging oxygen atom in key is replaced as the nucleotide groups of sulphur atom.
The modification of ribose groups refer to 2 ' in ribose groups-modification of hydroxyl (2 '-OH).In 2 '-hydroxyl of ribose groups
After base location introduces certain substituent groups such as methoxyl group or fluorine, ribalgilase is made to be not easy to cut nucleic acid, thereby increases nucleic acid
Stability makes nucleic acid have the stronger performance for resisting nuclease hydrolysis.The modification of 2 '-hydroxyl in nucleotide pentose is included
2 '-fluorine modification (2 '-fluro modification), 2 '-methoxyl group modification (2 '-OME), 2 '-methoxyethyl modification (2 '-
MOE), 2 ' -2,4- dinitrophenol modification (2 '-DNP modification), lock nucleic acid modification (LNA modification),
2 '-amido modified (2 '-Amino modification), 2 '-deoxidation modification (2 '-Deoxy modification) etc..
In situations where it is preferred, the antisense oligonucleotides contains the nucleotide groups of at least one modification, the modification
Nucleotide groups for thio-modification nucleotide groups and/or lock nucleic acid modification nucleotide groups；It is it is highly preferred that described anti-
Oligonucleotide has SEQ ID NO：Nucleotide sequence shown in 2, and the nucleotidyl at 1-5 and 17-21, wherein 5 ' ends
Phosphate group in group is by thio-modification.
The present invention is it should be pointed out that with the complete of characteristic as above or non-fully complementary RNA also the present invention's
In protection domain.Consider stability in the cell, the present invention preferably antisense oligonucleotides is DNA.
3) RNAi reagents
It is well known in the art that RNA interference (RNA interference, RNAi) is by double-stranded RNA (double-
Stranded RNA, dsRNA) induce, homologous mRNA efficient selective degradation the phenomenon that.It can be special due to the use of RNAi technology
The expression of specific gene is rejected or closed to the opposite sex, thus the technology have been widely used for exploring gene function and communicable disease and
The therapy field of malignant tumour.
RNAi reagents can be siRNA molecule, and typically one can theoretically form bobby pin (small
Hairpin) the single-stranded deoxy-oligonucleotide (shRNA) of structure, length do not exceed 100 nucleotide generally, typically not
Can be more than 75 nucleotide；The either double-strand deoxy-oligonucleotide (siRNA) of a 15-30bp, most typically 20-
23bp。
In some applications, RNAi reagents can also be the template DNA for encoding shRNA or siRNA.These template DNAs
It is likely to be present in carrier, such as the carriers such as plasmid vector or viral vectors；Can not also exist in carrier, only compiling for one section
The template DNA of code shRNA or siRNA adds a common promoter sequence segment that it is controlled to transcribe.
In the present invention, the miR-6069 inhibitor is the siRNA of miR-6069；Preferably, the small interference
The nucleotide groups that RNA contains at least one modification (modification mode is referred to antisense oligonucleotides acid moieties)；More preferably institute
State nucleotide groups and/or short peptide modified nucleotide groups of the nucleotide groups of modification for dimethoxy modification.
4)Inhibit nucleotide of promoter activity and the like
MiR-6069 inhibitor of the present invention can also include inhibiting the nucleotide of the promoter activity of miR-6069
And the like.Specifically, described nucleotide of promoter activity for inhibiting miR-6069 and the like can be with miR-
6069 promoter is combined, and inhibits its promoter activity, for example, morpholinyl (morpholino) modification nucleotide and its
Analog.
The third aspect, the present invention also provides a kind of function of inhibiting miR-6069 and/or the target bases for promoting miR-6069
The method of the expression of cause, this method include：MiR-6069 inhibitor is contacted with expressing the target cell of miR-6069, wherein, institute
The concrete type for stating miR-6069 inhibitor can be with as described above, in order to avoid unnecessary repetition, in this not go into detail.
Preferably, the target gene of the miR-6069 for PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1,
In FBXO31, CIDEB, TMEM127, SKI and PLEKHO1 at least two, at least three kinds, at least four, at least five kinds, at least
Six kinds, at least seven kinds, at least eight kinds, at least nine kinds, at least ten kinds, at least ten one kind or 12 kinds.
In the present invention, when the miR-6069 inhibitor is above-mentioned antisense oligonucleotides, due to the antisense widow core
Thuja acid can (complete complementary or partial complementarity) complementary with miR-6069, therefore, when the antisense oligonucleotides in vivo or body
When being contacted outside with expressing the target cell of miR-6069, the antisense oligonucleotides can carry out complementary pairing with miR-6069, and
Inhibit the combination of miR-6069 and its target gene (that is, inhibiting the activity of miR-6069), so as to break miR-6069 to it
The silence of target gene.
In the present invention, the method includes a effective amount of and miR-6069 complementations antisense oligonucleotides is introduced into table
Up in the target cell of miR-6069.Wherein, described " effective quantity " according to expression miR-6069 target cell difference and not
Together, and certain dosage effect is showed, those skilled in the art are according to conventional laboratory facilities and the expection reached
Purpose can readily determine the effective quantity of the target cell for expression miR-6069.
It, can be by way of conventional nucleic acid administration by antisense widow's core of the present invention when the contact is contact in vivo
Thuja acid is administered in individual.It is, for example, possible to use such a way carries out the administration of the antisense oligonucleotides：The antisense is few
Nucleotide can be administered or can also be noted by jet stream by way of virus infection, microinjection or Vesicle fusion
The mode penetrated is used for the intramuscular delivery of the antisense oligonucleotides.Alternatively, it is also possible to which the antisense oligonucleotides is coated to gold
On particle, then percutaneous dosing is carried out by the known manners such as particle bombardment equipment or " particle gun ".These are that this field is normal
The technological means of rule, this is no longer going to repeat them by the present invention.
Furthermore the target that the antisense oligonucleotides can also be introduced into expression miR-6069 in a manner of expression vector is thin
In born of the same parents.This kind of expression vector has the convenience restriction site positioned at promotor-proximal sequence in order to the antisense oligonucleotides
Insertion.Wherein, the transcription box in the expression vector can include transcription initiation region, target gene or its segment and transcription
Terminator.The carrier for example can be but be not limited to, plasmid, and virus etc., those skilled in the art can be according to reality
Situation is voluntarily selected.
In addition, the antisense oligonucleotides can also be introduced in expression by way of respiratory tract spray delivery
In the target cell of miR-6069, such as it is administered by way of being prepared into spray formulation.
In addition, the mode that the antisense oligonucleotides can also be administered orally is so as to be introduced in expression miR-6069
Target cell in, such as be administered by way of being prepared into oral preparation or by by the antisense oligonucleotides and food
The mode of mixing is administered orally.
It, can be by by the antisense oligonucleotides or containing the antisense oligonucleotides when the contact is vitro exposure
The carrier (for example, drug containing the antisense oligonucleotides) of acid, which is added directly into culture, the target cell of expression miR-6069
Matrix in contacted, and to being imported with the expression miR- of the antisense oligonucleotides under conventional cell culture condition
6069 target cell is cultivated.
In the present invention, when the miR-6069 inhibitor is above-mentioned RNAi reagents, the method includes by effective quantity
MiR-6069 siRNA be introduced into expression miR-6069 target cell in.The RNAi reagents and expression miR-6069
The contact of target cell may be internal contact or vitro exposure.The effective quantity and medication of the RNAi reagents can be with
With reference to as above to the description of antisense oligonucleotides progress, in order to avoid unnecessary repetition, in this not go into detail by the present invention.
Fourth aspect, the present invention also provides a kind of pharmaceutical composition, wherein, which contains above-mentioned miR-
6069 inhibitor and pharmaceutically acceptable carrier.
In the pharmaceutical composition of the present invention, the content of miR-6069 inhibitor as described above as active component can
To change in the larger context, for example, can be 0.001-99.99 weight %, preferably 0.01-99 weight %, more preferably
For 1-70 weight %, further preferably 5-30 weight %.
In the present invention, described pharmaceutical composition can be prepared as the various dosage forms of this field routine, the present invention to this simultaneously
It is not particularly limited, for example, solid can be configured to, semisolid, liquid or gas form, for example, tablet, capsule, the wine made of broomcorn millet
Agent, suspension, syrup, powder, particle, ointment, suppository, injection, inhalant, aerosol etc., the present invention be not another herein
One enumerates.
Therefore, the administrations of diversified forms can also be carried out according to the difference of pharmaceutical dosage form, such as, but not limited to, take orally to
Medicine, buccal administration, rectally, parenteral, Intraperitoneal medication, respiratory tract inhalation, intradermal administration, percutaneous dosing.
Wherein, the pharmaceutically acceptable carrier can carry out different selections according to the difference of dosage form, these are
It is known in those skilled in the art.Such as, but not limited to, the pharmaceutically acceptable carrier can be starch, colloid,
Lactose, glucose, sucrose, microcrystalline cellulose, kaolin, mannitol, calcium monohydrogen phosphate, sodium chloride, alginic acid etc..
Furthermore it is also possible to add in conventional additive such as solubilizer, isotonic agent, suspending agent, emulsifier, stabilizer and anti-corrosion
Agent.
In addition, the pharmaceutically acceptable carrier can also be specific including that can improve the antisense oligonucleotides targeting
Organ or tissue or the targeting agent of cell, the targeting agent for example can be targeting peptides, can also include that institute can be carried
Antisense oligonucleotides is stated to be easier to wear membrane reagent, such as cell-penetrating peptide, liposome, micro-capsule into the target cell of expression miR-6069
Bubble and membrane lipoprotein etc..
In the present invention, flavoring agent can also be added in described pharmaceutical composition, for example, peppermint, wintergreen etc..Separately
Outside, colorant can also be added in described pharmaceutical composition so that prepared dosage form has certain attraction in appearance
Power is distinguished with other products.
In the present invention, the antisense oligonucleotides can also can play the conventional medicine progress of similar effect with other
Combine to be prepared into combined medicinal composition.
5th aspect, the present invention also provides a kind of preventions and/or the method for the treatment of cancer, this method to include：Inhibit (by
Examination person) miR-6069 function and/or promote miR-6069 target gene expression；Or by above-mentioned miR-6069 inhibitor
And/or aforementioned pharmaceutical compositions are administered to subject.
In the present invention, the effective quantity of the administration, mode and dosage form are as described above, details are not described herein.
In situations where it is preferred, the subject in liver cancer subject, lung cancer subject and cutaneum carcinoma subject extremely
Few one；It is highly preferred that the target gene of the internal expression miR-6069 and/or miR-6069 of the subject；Further preferably
Ground, in the internal of the subject, the target gene of the excessively high expression of miR-6069 and/or miR-6069 cross low expression；Further
Preferably, the target gene of the miR-6069 for PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1,
In FBXO31, CIDEB, TMEM127, SKI and PLEKHO1 at least two, at least three kinds, at least four, at least five kinds, at least
Six kinds, at least seven kinds, at least eight kinds, at least nine kinds, at least ten kinds, at least ten one kind or 12 kinds.
6th aspect, the present invention also provides above-mentioned miR-6069 inhibitor, above-mentioned inhibition miR-6069 function and/or
The method of the method for expression of the target gene of miR-6069, aforementioned pharmaceutical compositions, above-mentioned prevention and/or treating cancer is promoted to exist
Application in the expression of the function of inhibiting miR-6069 and/or the target gene for promoting miR-6069；Particularly preventing and/or controlling
Treat the application in cancer and the disease similar to its symptom；Preferably, the cancer in liver cancer, lung cancer and cutaneum carcinoma at least
It is a kind of；It is highly preferred that the target gene of the miR-6069 for PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4,
At least two, at least three kinds, at least four, at least five in SASH1, FBXO31, CIDEB, TMEM127, SKI and PLEKHO1
Kind, at least at least six kinds, at least seven kinds, at least eight kinds, at least nine kinds, ten kinds, at least ten one kind or 12 kinds.
7th aspect, the present invention also provides above-mentioned miR-6069 inhibitor, aforementioned pharmaceutical compositions to prepare for pressing down
Application in the drug of the expression of the function of miR-6069 processed and/or the target gene of promotion miR-6069；Particularly preparing use
Application in prevention and/or the drug for the treatment of cancer and the disease similar to its symptom；Preferably, the cancer for liver cancer,
At least one of lung cancer and cutaneum carcinoma；It is highly preferred that the target gene of the miR6069 for RAMD4, BIRC3, BID,
At least two in TNFSF15, FOXO3, PPM1F and BCAP29.
In the present invention, the treatment refers to subject and the disease as caused by miR-6069 or the relevant symptom of state
Improve or completely disappear, wherein, the improvement on wide significance refers to reduce or increases at least one parameter.Specific to this Shen
Please, for example, can for target gene PPM1F, TNFSF15 of miR-6069, BCAP29, HRK, BID, GRAMD4, SASH1,
In FBXO31, CIDEB, TMEM127, SKI and PLEKHO1 at least two, at least three kinds, at least four, at least five kinds, at least
Six kinds, at least seven kinds, at least eight kinds, at least nine kinds, at least ten kinds, at least ten it is a kind of or 12 kinds expression raising.
The present invention will be described in detail by way of examples below.
Embodiment 1
The present embodiment is used for external tune of the antisense oligonucleotides to cancer cell for illustrating miR-6069 provided by the invention
Section acts on
Entrust the antisense oligonucleotides (miR- of the English dimension fine horse outstanding person artificial chemical synthesis miR-6069 of (Invitrogen) company
6069ASO, such as SEQ ID NO：Shown in 2) and commission Ji Ma companies synthesis Polo samples kinases 1 (PKL1) siRNA (positive-sense strands：
Such as SEQ ID NO：Shown in 4, antisense strand：Such as SEQ ID NO：Shown in 5).
(1) effect of the antisense oligonucleotides of miR-6069 to liver cancer cells
Use miR-6069 in quantitative PCR method detection liver cancer cells HuH-7 (being purchased from ATCC) and HepG2 (being purchased from ATCC)
Expression.Use the total serum IgE of Trizol extracting HuH-7 cells, HepG2 cells and normal primary human liver cell, Ran Houqu
The RNA of 1ug is inverted with Catch-All miRNA＆mRNA quantitative PCR kits (Kunshan Peng Ji Kai Feng bio tech ltd)
CDNA is recorded into, then by specification carries out quantitative PCR detection.The result shows that compared with normal primary human liver cell (control),
MiR-6069 expresses higher in HuH-7 and HepG2 cells.
Liver cancer cells HuH-7 and HepG2 are seeded in respectively in 384 well culture plates, cultivates and contains 10% tire ox in 50 μ L
In the DMEM culture mediums of serum.Cell incubator it is constant keep 37 DEG C and 5% CO2.With Lipofectamine 2000
(Invitrogen) siRNA (positive controls) or stochastic ordering of the PKL1 of 1 μM of miR-6069ASO or 40nM are transfected respectively
Arrange (negative control group, such as SEQ ID NO：Shown in 6), separately there is one group of control wells (transfection reagent group) only to add transfection reagent.
Cell is fixed with 4% paraformaldehyde (PFA) after transfecting 72 hours, and cell count is carried out after contaminating nucleus using DAPI.Knot
Fruit as depicted in figs. 1 and 2, wherein, data are represented with mean+SD, n=3, * * * P<0.001, * * P<0.01.
It can be seen from Fig. 1 and 2 compared with negative control group, the siRNA (positive control) and miR-6069ASO of PKL1
Can significantly reduce the number of HuH-7 and HepG2 cells；Also, compared with the siRNA of PKL1, miR-6069ASO makes
The number of HuH-7 and HepG2 cells reduces more.
(2) effect of the antisense oligonucleotides of miR-6069 to lung carcinoma cell
It is carried out according to the method for as above step (1), the difference is that replacing walking using lung cell A549 (being purchased from ATCC)
Suddenly the liver cancer cells HuH-7 and HepG2 used in (1).
Quantitative PCR the result shows that, compared with normal primary human pneumonocyte's (control), miR-6069 tables in A549 cells
Up to higher.
The result (as shown in Figure 3) of cell count shows compared with negative control group, the siRNA (positive control) of PKL1
Can significantly reduce the number of A549 cells with miR-6069ASO；Also, compared with the siRNA of PKL1, miR-
6069ASO makes the number of A549 cells reduce more.
(3) antisense oligonucleotides of miR-6069 has melanoma cells same inhibiting effect
It is carried out according to the method for as above step (1), the difference is that (being purchased from using melanoma cells SK-MEL-28
ATCC) the liver cancer cells HuH-7 and HepG2 instead of being used in step (1).
Quantitative PCR the result shows that, compared with normal primary human dermal's cell (control), miR-6069 is in SK-MEL-28
Expression in cell is higher.
Cell count the result shows that, compared with negative control group, the siRNA (positive control) and miR- of PKL1
6069ASO can significantly reduce the number of SK-MEL-28 cells；Also, compared with the siRNA of PKL1, miR-6069ASO
The number of SK-MEL-28 cells is made to reduce more.
(4) effect of the antisense oligonucleotides of miR-6069 to normal human fibroblasts
It is carried out according to the method for as above step (1), the difference is that (being purchased from using the thin HS27 of normal human desmocyte
ATCC) the liver cancer cells HuH-7 and HepG2 instead of being used in step (1).
The result (as shown in Figure 4) of cell count shows that compared with negative control group miR-6069ASO is to normal people
Fibroblastic growth has no significant effect.
Embodiment 2
The present embodiment is used to illustrate that the target gene of miR-6069 is lowered in liver cancer cells
Using TargetScan algorithms prediction miR-6069 target gene, and filter out with Apoptosis, growth inhibition or
The relevant gene of histone deacetylation, as shown in table 1.HepG2 liver cancer cells and normal primary human liver cell Trizol
It cracks (Invitrogen), then by specification extracts total serum IgE.With the water dissolution RNA of nuclease free, bioanalysis is then used
Instrument (bioanalyzer) detects the integrality of RNA, and the concentration of RNA is measured with Qubit3.TruSeq Stranded are used later
MRNA Sample Prep Kit (Illumina/USA) construction cDNAs library, and on 2500 sequenators of Hiseq (Illumina)
Sequencing.Obtain expressing one group of gene of downward in HepG2 cells through analysis, comparing discovery with the target gene of miR-6069 has 21
A gene for promoting apoptosis and the gene of 6 tumor suppressor genes downwards and 3 negative regulator AKT and TGF-beta accesses, as a result such as table 1
It is shown.
Table 1
Embodiment 3
The present embodiment is used to illustrate miR-6069 by the binding site on the UTR directly in conjunction with target gene with negative regulator target
The expression of gene
Respectively by target gene PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1, FBXO31, CIDEB,
The sequence of the non-translational region (UTR) containing miR-6069 binding sites of TMEM127, SKI and PLEKHO1 is (respectively such as SEQ ID
NO：7-18) directly synthesis is loaded to fiery luciferase (Fire luciferase) the gene 3' downstreams of pGL3-SV40 carriers
On xbaI sites, obtain above-mentioned each target gene miR-6069 experience carrier (commission Suzhou Hong Xun bio tech ltd into
Row).
By human embryonic kidney cells HEK-293T (being purchased from ATCC) culture in the DMEM culture mediums containing 10% fetal calf serum.Carefully
Born of the same parents' incubator it is constant keep 37 DEG C and 5% CO2.With the inoculum concentration of every 100,000 cells in hole by HEK-293T cell inoculations to 24
In porocyte culture plates, volume of culture is 500 μ L.Second day will be as follows with liposome 2000 (Invitrogen) to specifications
(Promega) is tested in the configuration reagent cotransfection to HEK-293T cells of table 2, after 36 hours with dual-luciferase assay to measure
The vigor for the luciferase expressed in carrier is experienced from miR-6069.Three repeating holes of setting every time, experiment is in triplicate.
Wherein, in each group, miR-6069 experiences the amount of being transferred to of carrier in terms of every hole：MiR-6069 experiences carrier 500ng,
miR-6069(SEQ ID NO：1, synthesized by Kunshan Peng Ji Kai Feng bio tech ltd) 1pmol, control RNA (Con
RNA, Ji Ma company synthesize, SEQ ID NO：19)1pmol.
Table 2
The results are shown in Figure 5.As seen in Figure 5, miR-6069 by be incorporated in target gene PPM1F, TNFSF15,
Bound site on the UTR of BCAP29, HRK, BID, GRAMD4, SASH1, FBXO31, CIDEB, TMEM127, SKI and PLEKHO1
The expression to inhibit target gene is put, wherein, * * * P<0.001.**P<0.01.
Embodiment 4
The present embodiment is used for the miR-6069's for the antisense oligonucleotides and mispairing for illustrating miR-6069 provided by the invention
Antisense oligonucleotides can inhibit the function of miR-6069
By human embryonic kidney cells HEK-293T cultures in the DMEM culture mediums containing 10% fetal calf serum.Cell incubator is permanent
Surely keep 37 DEG C and 5% CO2.With the inoculum concentration of every 100,000 cells in hole by HEK-293T cell inoculations to 24 hole cell culture
In plate, volume of culture is 500 μ L.Second day with liposome 2000 (Invitrogen) to specifications by the configuration of such as the following table 3
(Promega) is tested in reagent cotransfection to HEK-293T cells, after 36 hours with dual-luciferase assay to measure from miR-
6069 experience the vigor for the luciferase expressed in carrier.Three repeating holes of setting every time, experiment is in triplicate.
Wherein, in each group, miR-6069 experiences the amount of being transferred to of carrier and nucleotide in terms of every hole：MiR-6069 impressions carry
Body (PPM1F UTR) 500ng, miR-6069 (such as SEQ ID NO：Shown in 1, by Kunshan Peng Ji Kai Feng bio tech ltd
Synthesis) 1pmol, control nucleotide (Con RNA, SEQ ID NO：Shown in 19 and Con DNA, such as SEQ ID NO：Shown in 20)
1pmol, miR-6069ASO 1pmol, miR-6069ASO (such as SEQ ID NO of mispairing：Shown in 3) 1pmol.
The results are shown in Figure 6.As seen in Figure 6, miR-6069ASO can completely inhibit the function of miR-6069,
But the nucleotide of random controls cannot inhibit the function of miR-6069.And the miR-6069ASO containing base mismatch also can
Part inhibits the function of miR-6069.***P<0.001, * * P<0.01.
Table 3
Embodiment 5
The present embodiment is used to illustrate that the antisense oligonucleotides of the miR-6069 of thio-modification provided by the invention is thin to cancer
The external adjustment effect of born of the same parents
The procedure of Example 1 was followed except that using the 1-2 positions and 19-20 at 5 ' ends by thio-modification
Antisense oligonucleotides (such as SEQ ID NO of miR-6069：Shown in 2) replace unmodified miR- used in embodiment 1
6069 antisense oligonucleotides (miR-6069ASO, such as SEQ ID NO：Shown in 2).
The result (as shown in Figure 7) of cell count shows compared with unmodified miR-6069ASO, thio-modification
It is more that miR-6069ASO reduces the number of liver cancer cells HuH-7.
By the result of above example 1-5 it is found that the present invention is by by inhibitor (such as miR- of miR-6069
6069ASO, the miR-6069ASO of mispairing, thio-modification miR-6069ASO) target cell (such as liver with high expression miR-6069
Cancer cell, lung carcinoma cell, melanoma cells etc.) it is contacted, it can fully inhibit the function of miR-6069 and promote its target
Gene (PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1, FBXO31, CIDEB, TMEM127, SKI and
PLEKHO1 expression).This explanation can effectively prevent when by the inhibitor for individual administration and/or treat miR-
6069 high expression and/or its target gene (PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1, FBXO31,
At least two in CIDEB, TMEM127, SKI and PLEKHO1) cross low expression caused by disease, for example, various cancers or with
The similar disease of its symptom, particularly at least one of liver cancer, lung cancer and cutaneum carcinoma.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, it can be combined by any suitable means.In order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
SEQUENCE LISTING
<110>Kunshan Peng Ji Kai Feng bio tech ltd
<120>Pass through the method and drug of miR-6069 progress anticancers and its application
<130> I42840PCG
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> RNA
<213> Human
<400> 1
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<210> 2
<211> 21
<212> DNA
<213> Artificial
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<223> The sequence is synthesized.
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<213> Artificial
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<223> The sequence is synthesized.
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Claims (9)
1. the expression of the function of inhibiting miR-6069 and/or the target gene for promoting miR-6069 in prevention and/or treating cancer and
Application in the disease similar to its symptom, particularly preparing for preventing and/or treating cancer and similar to its symptom
Application in the drug and/or food of disease；
Preferably, the cancer is at least one of liver cancer, lung cancer and cutaneum carcinoma；
Preferably, miR-6069 has SEQ ID NO：Nucleotide sequence shown in 1；
Preferably, the target gene of the miR-6069 for PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1,
At least two in FBXO31, CIDEB, TMEM127, SKI and PLEKHO1.
2. a kind of miR-6069 inhibitor, which is characterized in that the miR-6069 inhibitor is the antisense oligonucleotides of miR-6069
Nucleotide of promoter activity of acid, the siRNA of miR-6069 and inhibition miR-6069 and the like；
Preferably, the antisense oligonucleotides includes antisense DNA and antisense RNA；
Preferably, the antisense oligonucleotides and miR-6069 are complementary, and with the length of 12-30 nucleotide, and have
At least with the sequence of the 1-8 positions nucleotide complementations at the 5 ' of miR-6069 ends.
3. miR-6069 inhibitor according to claim 2, wherein, the antisense oligonucleotides has following nucleotide
Sequence：
a)SEQ ID NO：Nucleotide sequence shown in 2；
B) in SEQ ID NO：In nucleotide sequence shown in 2 through lack, replace or add one or several nucleotide and with
The nucleotide sequence of miR-6069 complete complementaries or partial complementarity；
Preferably, the antisense oligonucleotides has in SEQ ID NO：Through lacking, replacing or add in nucleotide sequence shown in 2
Add one or several nucleotide and at least there is 60% complementary nucleotide sequence with miR-6069；
Preferably, the 1-8 positions nucleotide at the 5 ' ends of the antisense oligonucleotides and miR-6069 at most has the mistake of 2 nucleotide
The nucleotide sequence matched；
Preferably, the antisense oligonucleotides has SEQ ID NO：Nucleotide sequence shown in 3；
Preferably, the antisense oligonucleotides contains the nucleotide groups of at least one modification, the nucleotide groups of the modification
The nucleotide groups that nucleotide groups and/or lock nucleic acid for thio-modification are modified；
Preferably, the antisense oligonucleotides has SEQ ID NO：Nucleotide sequence shown in 2, and wherein 5 ' end 1-5 with
The nucleotide groups of 17-21 are by thio-modification.
4. miR-6069 inhibitor according to claim 2, wherein, the miR-6069 inhibitor is the small of miR-6069
RNA interfering；
Preferably, the siRNA contains the nucleotide groups of at least one modification；
Preferably, nucleotide groups and/or short peptide modified nucleosides of the nucleotide groups of the modification for dimethoxy modification
Acid groups.
5. a kind of method of the expression of function of inhibiting miR-6069 and/or the target gene for promoting miR-6069, which is characterized in that
This method includes：MiR-6069 inhibitor is contacted with expressing the target cell of miR-6069, wherein, the miR-6069 inhibitor
For the miR-6069 inhibitor described in any one in claim 2-4；
Preferably, the target gene of the miR-6069 for PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1,
At least two in FBXO31, CIDEB, TMEM127, SKI and PLEKHO1.
6. a kind of pharmaceutical composition, which is characterized in that the pharmaceutical composition contains in claim 2-4 described in any one
MiR-6069 inhibitor and pharmaceutically acceptable carrier.
7. a kind of prevention and/or the method for the treatment of cancer, which is characterized in that this method includes：
The expression of the function of inhibiting miR-6069 and/or the target gene for promoting miR-6069；Or
By the miR-6069 inhibitor described in any one in claim 2-4 and/or the pharmaceutical composition described in claim 6
It is administered to subject；
Preferably, the subject is at least one of liver cancer subject, lung cancer subject and cutaneum carcinoma subject；
Preferably, the target gene of the internal expression miR-6069 and/or expression miR-6069 of the subject；
Preferably, in the internal of the subject, miR-6069 high is expressed and/or the target gene low expression of miR-6069；
Preferably, the target gene of the miR-6069 for PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1,
At least two in FBXO31, CIDEB, TMEM127, SKI and PLEKHO1.
8. the miR-6069 inhibitor described in any one, the method described in claim 5, claim 6 in claim 2-4
Method described in the pharmaceutical composition, claim 7 is inhibiting the function of miR-6069 and/or is promoting the target of miR-6069
Application in the expression of gene；Application particularly in prevention and/or treating cancer and the disease similar to its symptom；It is preferred that
Ground, the cancer are at least one of liver cancer, lung cancer and cutaneum carcinoma；It is highly preferred that the target gene of the miR-6069 is
In PPM1F, TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1, FBXO31, CIDEB, TMEM127, SKI and PLEKHO1
At least two.
9. the miR-6069 inhibitor described in any one and/or the pharmaceutical composition described in claim 6 in claim 2-4
Application in the drug of the expression of the function of preparing for inhibiting miR-6069 and/or the target gene for promoting miR-6069；It is special
It is not to prepare for the application in prevention and/or the drug for the treatment of cancer and the disease similar to its symptom；Preferably, it is described
Cancer is at least one of liver cancer, lung cancer and cutaneum carcinoma；It is highly preferred that the target gene of the miR-6069 for PPM1F,
In TNFSF15, BCAP29, HRK, BID, GRAMD4, SASH1, FBXO31, CIDEB, TMEM127, SKI and PLEKHO1 at least
Two kinds.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015100257A1 (en) * | 2013-12-23 | 2015-07-02 | The General Hospital Corporation | Methods and assays for determining reduced brca1 pathway function in a cancer cell |
| CN104955950A (en) * | 2012-09-26 | 2015-09-30 | 米尔克斯治疗学公司 | Oligomers with improved off-target profile |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104955950A (en) * | 2012-09-26 | 2015-09-30 | 米尔克斯治疗学公司 | Oligomers with improved off-target profile |
| WO2015100257A1 (en) * | 2013-12-23 | 2015-07-02 | The General Hospital Corporation | Methods and assays for determining reduced brca1 pathway function in a cancer cell |
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