CN101121934B

CN101121934B - Multiple target points miRNA antisense nucleic acid technique

Info

Publication number: CN101121934B
Application number: CN2007100720248A
Authority: CN
Inventors: 杨宝峰; 王志国; 吕延杰; 白云龙; 初文峰
Original assignee: Harbin Medical University
Current assignee: Harbin Medical University
Priority date: 2007-04-11
Filing date: 2007-04-11
Publication date: 2011-12-07
Anticipated expiration: 2027-04-11
Also published as: CN101121934A

Abstract

The present invention discloses a preparation method of multi-target miRNA antisense nucleotide, relating to an antisense nucleotide technology. The present invention is capable to solve the problems of existing method for silencing miRNAs, e.g. single target and poor action efficacy. The preparation method of multi-target miRNA antisense nucleotide is achieved by the following procedures: 1. miRNA that capable to regulate the key gene (i.e. pathogenic gene) is found through gene chip screening or provision of documentation; 2. a plurality of antisense miRNA antisense nucleotides are designed into a single-nucleotide sequence that can silence a plurality of multi-target miRNAs, i.e. the preparation method of multi-target miRNA antisense nucleotide is achieved. The present invention can be used as a simple and direct method to conduct study on the bioprocess containing various miRNAs and genes. Compared with existing methods, the greatest advantage of CAMO method lies in that one drug molecule is capable to regulate multi-target genes and the action efficacy is stronger. The preparation method of multi-target miRNA antisense nucleotide provided by the present invention can well settle the problems of existing methods, i.e. single target and poor action efficacy.

Description

The preparation method of multiple target points miRNA antisense nucleic acid

Technical field

The present invention relates to a kind of antisense nucleic acid technique.

Background technology

Thereby antisense nucleotide is RNA or dna molecular that one section and mRNA or the pairing of DNA specificity combine the synthetic of blocking its genetic expression.Antisense nucleotide can or suppress tumour cell and come specificity to suppress duplicating of cell proliferation and virus with viral crucial encoding gene by sealing, is the newtype drug for the treatment of tumour, virus disease and other relative disease.Kind surplus the antisense nucleotide medicine of studying at present has 10, wherein antitumor action has 7 kinds, a kind of antivirus action, other is cardiovascular as acting on, metabolism, immunity and cell adhesion system have 4 kinds.The generation of disease, the result of several often or a plurality of gene comprehensive actions, these antisense nucleotide medicinal designs at present all are to design at the pass key sequence that certain single Disease-causing gene or certain are regulated virogene protein expression (enzyme).The discovery of microRNA s (miRNAs) has changed us to regulating the understanding of genetic expression mechanism.Illustrate that according to the quantity (estimate content＞1% in Human genome, regulate 1～10% gene) and the expression level (copy number＞1000 of some miRNAs in each cell) of miRNAs in genome miRNAs is a kind of content rich RNA kind in human body.And each miRNA has the potential of regulating hundreds of mRNAs; Therefore miRNAs has function widely in cell.At present proved that miRNAs is the new treatment target spots of these diseases in the series of human disease that comprises cancer.Therefore, the valid approach of reticent miRNAs is to utilize the antisense nucleotide of miRNA.Design the antisense nucleotide medicine targetedly according to miRNAs changes of function in the disease and intervene, this is a kind of basic approach in miRNAs research and miRNAs treatment.The combination of these miRNA antisense nucleotide has specificity, dose-dependently, validity and non-reversibility.This method has been used to identify many cytology functions of miRNAs, and people have believed that it is a kind of rational countermeasure of treatment disease.The main method of present reticent miRNAs is Anti-miRNA antisense inhibitor (Anti-miRNA antisense inhibitors or anti-miRNA oligonucleotides, AMO) method, but this method exists target single, and the problem of difference is renderd a service in effect.

Summary of the invention

The present invention is that the problem of difference is renderd a service in effect for the method that solves existing reticent miRNAs exists target single, and has proposed the preparation method of multiple target points miRNA antisense nucleic acid.The preparation method of multiple target points miRNA antisense nucleic acid realizes by the following method: one, provide by gene chip screening or document, find the miRNA that can regulate key gene; Two, according to selected a plurality of target miRNA, the selected target miRNA of synthetic antisense nucleotide chain separately is called the antisense unit; Three, with the antisense unit design to of a different target miRNA energy simultaneously in the single-nucleotide sequence of reticent a plurality of target miRNAs, the antisense unit that promptly connects two different target miRNA with the chain of eight Nucleotide, wherein the two ends of the chain of eight Nucleotide are connected to unitary 5 ' end of antisense and the 3 ' end of two different target miRNA, five Nucleotide at two ends, antisense unit are locked by methylene bridge, the chain of eight Nucleotide is CTTAAATG, promptly realizes the method for multiple target points miRNA antisense nucleic acid.The present invention will be that a plurality of antisense nucleotide of target are incorporated in the single nucleotide fragments at different miRNA, this fragment has many targets property to miRNAs---compound miRNAs (combined anti-miRNA oligonucleotides, CAMO), it is a kind of improved route of seeking miRNAs target spot and research miRNAs function, and this mainly is because " single slice-many target spots " method of CAMO can be the unitary shaped body of antisense that carries the unitary phenogen of the same antisense of multiple or carry the different miRNA of multiple target.Because a specific mRNA is regulated by multiple miRNA, for the Optimal Jamming effect that obtains gene needs the multiple miRNA of target simultaneously, and the present invention can accomplish this point, and this is that the CAMO method is better than AMO method part.Because various objectives needs the combination of multiple different antisense unit sets, CAMO provides one to have the innovation and the method for design and rational for this reason, for a kind of instrument of precision is provided in the target spot confirming and differentiate miRNA and their functional analyses such as effect in the gene regulating process.The present invention can be used as a kind of simply and directly method, can study other bioprocess that comprises multiple miRNA and several genes.CAMO method its sharpest edges of comparing with existing method are that can make a drug molecule regulate a plurality of target genes, action effect is more remarkable, and it is single to have solved existing method target, and the problem of difference is renderd a service in effect.

Description of drawings

Fig. 1 a is that the binding sequence segment is embedded into the gene fragment figure on 3 ' UTR of the luciferase gene in the HEK293 cell, Fig. 1 b is the comparison diagram that the AMO of CAMO21/155/17 and rule eliminates retarding effect, and Fig. 1 c is the comparison diagram that the AMO of CAMO1/133 and rule eliminates retarding effect; Fig. 2 a imports CAMO21/155/17 and imports miR-21 respectively in human breast cancer cell MCF-7, the TGFBI of three kinds of segmental tumor suppressor genes of antisense nucleotide of miR-155 and miR-17-5p, the increase situation comparison diagram of APC and BCL2L11, Fig. 2 b is that the CAMO1/133 transfection increases the comparison diagram that HCN2 protein level and AMO1 and AMO133 cotransfection increase the HCN2 protein level in the newborn rat ventricular muscle cell that exsomatizes, Fig. 2 c is uciferase activity expression figure after elimination 3 ' UTRs and the miRNAs interaction result, Fig. 2 d uses behind the reticent HCN2 of CAMO1/133 and the CACNA1C gene 3 ' UTRs and uciferase activity expression figure after the miRNAs interaction result, Fig. 2 e be the present invention in MCF-7 clone the pair cell viability influence figure, Fig. 2 f is the target miRNAs results of interaction figure of CAMOs.

Embodiment

Embodiment one: the preparation method of multiple target points miRNA antisense nucleic acid realizes by the following method in the present embodiment: one, provide by gene chip screening or document, find the miRNA that can regulate key gene; Two, according to selected a plurality of target miRNA, the selected target miRNA of synthetic antisense nucleotide chain separately is called the antisense unit; Three, the antisense unit design to of a different target miRNA energy simultaneously in the single-nucleotide sequence of reticent a plurality of target miRNAs, is promptly realized the method for multiple target points miRNA antisense nucleic acid.

Embodiment two: the difference of present embodiment and embodiment one is that a plurality of of step 2 are 2～5.Other step is identical with embodiment one.

Embodiment three: present embodiment and the difference of embodiment one are in the step 3 antisense unit design to an energy of different target miRNA is realized in the single-nucleotide sequence of reticent a plurality of target miRNAs simultaneously by the following method: the antisense unit that is connected two different target miRNA with the chain of eight Nucleotide, wherein the two ends of the chain of eight Nucleotide are connected to the antisense unitary 5 ' and 3 ' of two different target miRNA, and five Nucleotide at two ends, antisense unit are locked by methylene bridge.Other step is identical with embodiment one.

Embodiment four: the difference of present embodiment and embodiment three is that the chain of eight Nucleotide is CTTAAATG.Other step is identical with embodiment one.

Embodiment five: present embodiment is enumerated the present invention of example proof, present embodiment realizes by the following method: present MiR-21, miR-155, be proved to be with miR-17-5p and be carcinogenic miRNAs, their overexpressions in some solid tumors, miR-1 and miR-133 are the miRNAs of decision muscle property, assay determination goes out them makes the ability of target miRNAs inactivation separately, with a binding sequence (miR-21 that 5 target miRNAs are arranged, miR-155, miR-17-5p, miR-1 and miR-133) segment is embedded on 3 ' UTR of the luciferase gene in the HEK293 cell.

5 target miRNAs binding sequence (miR-21 will be arranged in the present embodiment, miR-155, miR-17-5p, miR-1 and miR-133) segment is embedded on 3 ' UTR of the luciferase gene in the HEK293 cell and realizes by the following method: as shown in Figure 1a: connect the antisense unit at two ends with the link (underlining mark) of eight Nucleotide, five Nucleotide at two ends are locked by methylene bridge; The base of inserting in eight amino acid of each antisense unit (capitalization runic) 5 ' end is replaced sudden change (small letter runic) and the CAMOs of sudden change has been classified as negative control.Shown in Fig. 1 b: CAMO21/155/17 has eliminated miR-21 in the HEK293 cell, these three kinds of materials of miR-155 and miR-17-5p cause the retarding effect of uciferase activity, mutant CAMO21/155/17 (MTCAMO21/155/17) as negative control does not have this effect simultaneously, shown in Fig. 1 c: can observe similar result with CAMO1/133 yet.

Be the checking The above results, the selected gene that comprises the miRNA target spot with 3 ' UTR has been assessed the effect of these two CAMOs at protein level; Shown in Fig. 2 a: in human breast cancer cell MCF-7, import CAMO21/155/17 and import miR-21 respectively, three kinds of antisense nucleoside acid fragments of miR-155 and miR-17-5p are compared, CAMO21/155/17 has increased tumor suppressor gene TGFBI more significantly, the level of APC and BCL2L11.Shown in Fig. 2 b: in the newborn rat ventricular muscle cell that exsomatizes, the CAMO1/133 transfection has increased by the level of two kinds of ionophorous protein HCN2 (subunit of pacemaker passage) and Cav1.2 (alpha hypotype of the L type calcium channel of CACNA1C coding) significantly; 3 ' UTR of HCN2 comprises the supposition target spot of miR-1 and miR-133,3 ' the UTR of CACNA1C only comprises the target spot of a miR-133, obviously, the protein level of HCN2 produce significantly effect neither AMO1 neither AMO133, when a gene is regulated simultaneously by a plurality of miRNAs, service regulations AMOs can produce false negative result, AMO1 and AMO133 cotransfection also increase the protein level of HCN2, obviously reduce but compare degree, illustrate that the CAMO1/133 transfection increases the protein level height of HCN2 with CAMO1/133.

In order to verify TGFBI, APC and BCL2L11 are respectively miR-21 really, the homology target spot of miR-155 and miR-17-5p, and HCN2 and CACNA1C are the homology target spots of miR-1 and miR-133, and we include the 3 ' UTRs in miRNAs prediction site or 3 ' UTR that segment is inserted into the luciferase acceptor carrier to 3 ' UTRs from these genes.This chimeric vectors comprises TGFBI, APC and BCL2L113 ' UTRs, contain miR-21 simultaneously, miR-155 and miR-17-5p also contain AMOs, the CAMO21/155/17 of CAMO21/155/17 or sudden change, and gone in the HEK293 cell by cotransfection, shown in Fig. 2 c, that uciferase activity is expressed after elimination 3 ' UTRs and the miRNAs interaction result is the CAMO21/155/17 of CAMO21/155/17 rather than sudden change, yet none AMO reverses the expression of uciferase activity fully; Shown in Fig. 2 d: with the reticent HCN2 of CAMO1/133 and CACNA1C gene 3 ' UTRs, can observe similar uciferase activity expression of results, TGFBI is described, APC and BCL2L11 are respectively miR-21 really, the homology target spot of miR-155 and miR-17-5p, and HCN2 and CACNA1C are the homology target spots of miR-1 and miR-133.

CAMO21/155/17 is designed to fixed these three the highest carcinogenic miRNAs of overexpression frequency in many cancers of target, shown in Fig. 2 e: we observe the influence of its pair cell viability in MCF-7 clone, show that from the result of control group and MT CAMO21/155/17 group concentration is that 10nMCAMO21/155/17 makes survivaling cell reduce 1～18%, and must reach 100nM for the concentration that reaches same effect AMO.Use AMO-21, each 10nM of AMO-15 and AMO-17 (30nM altogether, AMO-mixture 1) existence of pair cell produces more significantly than 10nM CAMO21/155/17 and reduces or behind each 3.3nM (10nM, AMO-mixture 2) altogether cotransfection, illustrates that target of the present invention is fixed effective.

For the effect that proves CAMOs is their target miRNAs results of interaction really, we measure miRNAs, shown in Fig. 2 f: AMOs is by unknown mechanisms of degradation target miRNAs, CAMOs then reduces their target miRNAs significantly, and these CAMOs do not influence the mRNA level of their target gene, and the effect that CAMOs is described is their target miRNAs results of interaction really.

Claims

1. the preparation method of multiple target points miRNA antisense nucleic acid is characterized in that the preparation method of multiple target points miRNA antisense nucleic acid realizes by the following method: one, provide by gene chip screening or document, find the miRNA that can regulate key gene; Two, according to selected a plurality of target miRNA, the selected target miRNA of synthetic antisense nucleotide chain separately is called the antisense unit; Three, with the antisense unit design to of a different target miRNA energy simultaneously in the single-nucleotide sequence of reticent a plurality of target miRNAs, the antisense unit that promptly connects two different target miRNA with the chain of eight Nucleotide, wherein the two ends of the chain of eight Nucleotide are connected to unitary 5 ' end of antisense and the 3 ' end of two different target miRNA, five Nucleotide at two ends, antisense unit are locked by methylene bridge, the chain of eight Nucleotide is CTTAAATG, promptly realizes the method for multiple target points miRNA antisense nucleic acid.

2. method according to claim 1 is characterized in that a plurality of in the step 2 are 2～5.