WO2005017145A1 - Methode d'identification reelle ou presumee d'un gene soumis a une regulation commandee par un arn fonctionnel et utilisation de cette methode - Google Patents

Methode d'identification reelle ou presumee d'un gene soumis a une regulation commandee par un arn fonctionnel et utilisation de cette methode Download PDF

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WO2005017145A1
WO2005017145A1 PCT/JP2004/011624 JP2004011624W WO2005017145A1 WO 2005017145 A1 WO2005017145 A1 WO 2005017145A1 JP 2004011624 W JP2004011624 W JP 2004011624W WO 2005017145 A1 WO2005017145 A1 WO 2005017145A1
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gene
functional rna
controlled
region
expression
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PCT/JP2004/011624
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Japanese (ja)
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Roberto Antonio Barrero
Takuro c/o BITS Co. Ltd. TAMURA
Tadashi Imanishi
Takashi Gojobori
Kazunari Taira
Hiroaki Kawasaki
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Japan Biological Informatics Consortium
Bits Co., Ltd.
National Institute Of Advanced Industrial Science And Technology
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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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function

Definitions

  • the present invention relates to the field of biomolecule control. More specifically, the present invention relates to a method for controlling gene expression using a nucleic acid molecule.
  • RNAi Ribonucleic acid molecules regulate gene expression by RNAi in animals and in plants by RNAi.
  • Non-Patent Document 1 Hutvagner and Zamore 2002 Curr. Opin. Gen. Dev. 12: 225-232.
  • RNAi and PTGS inhibit gene expression by selectively destroying mRNAs that are highly complementary to siRNA sequences.
  • This method is used in the field of gene expression research to suppress the expression of a target gene by artificially introducing double-stranded RNA into cells.
  • a single-stranded short RNA called miRNA has been attracting attention as a short biological RNA molecule involved in the control of gene expression (Non-patent document 2 Lagos-Quintana et al.
  • miRNAs small 21-22 base RNA families in three groups, Drosophila melanogaster, nematodes, and humans. These small RNAs were considered specific at the time of development and were named MicroRNAs (miRNAs).
  • miRNAs One of the characteristics of miRNA is that when predicting the secondary structure of a sequence that is considered to be precursor RNA, the stem-loop structure shRNA (short hairpin RNA) containing all dsRNA (duplex RNA) regions is used. .
  • miRNAs are transcribed as precursor RNAs, dozens to hundreds of bases long with a basil structure containing base pair mismatches, presumably to prevent the expression of various genes induced by dsRNA. (Non-patent document 3 RNAi experimental protocol p.22).
  • Non-Patent Document 4 Eric C. Lai Nature Genetics 30, 363-364 (2002).
  • siRNAs inhibit gene expression by specifically cleaving target mRNAs with high complementarity.On the other hand, miRNAs are thought to act on mRNA with a 3 'UTR complementary region and suppress its translation.
  • Non-patent document l Hutvagner and Zamore 2002 Curr.Opin.Gen. Dev. 12: 225-232
  • Non-patent document 2 Lagos-Quintana et al. 2001 Science 294: 858-862
  • Non-Patent Document 3 “RNAi Experimental Protocol”, Separate Volume on Experimental Medicine, p.22 (2003)
  • Non-Patent Document 4 Eric C ⁇ ai Nature Genetics 30, 363-364 (2002)
  • RNAi and PTGS in living organisms are to function as an ecological defense system and to destroy foreign RNA contaminated by bacterial infection.
  • gene expression control using these is mainly intended for artificial control points.
  • miRNAs are thought to control gene expression by interacting with target mRNAs.
  • the regulation of mRNA expression by miRNA is a mechanism of gene regulation that exists in nature. If miRNA and one or more regulated genes that are targets of the miRNA can be predicted or identified, this mechanism regulates the regulated gene. It becomes possible to intentionally manipulate gene expression control in living organisms for genes.
  • the present invention as a method for predicting or identifying miRNAs and one or more controlled genes as their targets, it has been confirmed that they are present in human or other animal or plant cells, The characteristics of the base sequence of miRNA were examined for the target miRNA molecule, and the target mRNA molecule was predicted in consideration of the obtained characteristics.
  • the present invention provides a method for predicting or identifying a gene (mRNA) controlled by a target of a functional RNA molecule capable of controlling gene expression, for example, miRNA or the like, as a target of the functional RNA molecule. I do.
  • the present invention provides a method for controlling the expression of an mRNA using the miRNA by identifying the mRNA targeted by the miRNA, and the method of using the miRNA as an active ingredient for controlling the expression of the mRNA. And a drug containing the expression control agent.
  • the present invention also includes preparing an siRNA having a degrading effect on the miRNA, and releasing, in the cell, a controlled gene whose translation is inhibited by the miRNA from translation inhibition and translating the gene. included.
  • the functional RNA molecule of the present invention is an RNA molecule having a length of 16 to 25 bases.
  • the functional RNA molecule includes miRNA.
  • RNA molecule in the present invention a naturally occurring miRNA can be used for any living organism such as human, nematode, and fly fly.
  • RNA molecule is a miRNA
  • the method for predicting or identifying a gene controlled by a miRNA (regulated gene) of the present invention includes the following first step and second step.
  • the base sequence of the miRNA molecule is not shared with a region having a region common to a plurality of miRNAs (conserved region), and a region having a base sequence (region other than the conserved region) (Non-storage area).
  • ambiguity search accuracy level value set by a separate criterion is given to the conserved area with a relatively gentle criterion and to the non-conserved area with a relatively strict criterion.
  • This is a step (process) of searching for a gene (a controlled gene candidate) having a base sequence complementary to the search base sequence (functional RNA molecule) as the search base sequence.
  • a search for “regions complementary to the entire miRNA sequence” can be performed by requesting different degrees of complementarity between conserved and non-conserved regions.
  • the full-length functional RNA molecule sequence combining the conserved region and the non-conserved region is set to a relatively mild standard, the non-conserved region is set to a strict standard, and each is set to a different standard.
  • a step (step) of searching for a gene (candidate to be controlled) having a base sequence complementary to a functional RNA molecule can be used as a search base sequence.
  • controlled gene candidate having a search accuracy level or more is found in the second step, this is predicted or identified as a gene controlled by the functional RNA molecule (controlled gene).
  • any miRNA sequence already collected in various databases can be used.
  • a sequence complementary to the motif sequence among several miRNAs (having a common property) in a region having a length of 6 to 8 bases from the 5 'end, as compared with various miRNAs, Sequence) is found to be present.
  • Examples of motif sequences are shown in FIGS. 4 and 5.
  • Such motif sequences include, for example, UAUCACAG PowerS.
  • the 6-8 base region from the 5 'end of the miRNA is called a "conserved region", which is adjacent to the conserved region.
  • the 8 bases region from the 5 'end of the miRNA is classified as a "conserved region”
  • the 9 bases after the 5' end adjacent to the conserved region are classified as a "non-conserved region”.
  • the first base from the 5 'end of the miRNA may be different from the motif in some cases, at least the 5' end force and the 57 base region from the second base may be used as a conserved region. .
  • a certain miRNA is a target gene for regulation, that is, a candidate for a regulated gene. Since sequences of a large number of genes (cDNAs) are already stored in the database, cDNA sequences stored in any database such as DDBJ and EBI can be used. Particularly preferably, a cDNA sequence stored in a database of NCBI (US National Center for Biotechnology Information, hereinafter abbreviated as NCBI) can be used.
  • NCBI US National Center for Biotechnology Information
  • cDNA containing the 3 'UTR is extracted from any database. Then, for each of the extracted cDNAs, from the start position of the 3 ′ UTR, that is, immediately after the end of the protein coding region on the mRNA, for example, within a range of 3500 to 500 bases, specifically 3000 Bases, preferably 1500 bases, more preferably 1000 bases, can be the sequence under consideration.
  • the miRNA is compared with the sequence to be examined (candidate target base sequence), and a certain level (a relatively mild standard for the total length of the functional RNA molecule within the search accuracy level) is determined.
  • a candidate primary target sequence having a portion having a certain degree of complementarity with the miRNA sequence is first selected as a complementary region.
  • the complementarity is more than an appropriate value selected from 50-90%, preferably between 60% -75%, specifically, 50% or more, 55% or more , 80% or more, or 90% or more, preferably 60% or more, 65% or more, 70% or more, or 75% or more complementarity.
  • the primary selection is performed using a target base sequence 1J having a region in which two-thirds or more bases are complementary to the miRNA base sequence as a complementary region.
  • the phrase that two-thirds or more of the bases are complementary means that two-thirds or more of the number of bases of the miRNA is complementary.
  • the miRNA is composed of 22 bases, Means that at least 15 bases are complementary. Relatively mild criteria for the total length of a functional RNA molecule can employ such selection criteria.
  • the mismatch rate is changed in the storage area and the non-storage area so as to reduce the mismatch in the non-storage area.
  • the allowable mismatch rate is stored.
  • the non-conserved region is less than half, preferably less than one-fourth, and the gene whose base sequence satisfies the mismatch ratio between the two is controlled by the miRNA (controlled gene) It is also possible to adopt a method of predicting or identifying that
  • the prediction or identification of the method of the present invention may further include, for example, a step of confirming that the regulated gene identified in the second step is controlled by the miRNA.
  • the step of confirming that the miRNA controls and regulates the regulated gene is the step of actually introducing the miRNA into the sample cell to determine whether the expression of the predicted or identified regulated gene is affected.
  • a means for detecting the presence of mRNA or protein expression such as Northern blotting and Western blotting, can be used.
  • the effect on the expression of the controlled gene is preferably a fragment of the candidate controlled gene or its complementary strand, usually 15 or more bases in a continuous manner that can distinguish the candidate controlled gene or its complementary strand from other genes.
  • a DNA chip in which a large number of probe sequences of about 20 to 30 bases are arranged, the expression of miRNA-introduced cells can be confirmed for the collected mRNA or cDNA library.
  • a DNA chip microarray
  • the expression of the target gene can be controlled using the miRNA, and the miRNA can be used as an agent for controlling the expression of the target gene.
  • miRNA is an expression vector that produces a miRNA that can be directly introduced into cells. Can be prepared.
  • Target gene expression inhibitors include miRNAs or miRNA expression vectors and, if necessary, other additives effective for introduction into the target organism, such as calcium phosphate, ribofurin, polylysine, and other additives. Can be contained.
  • RNA expression vector For preparation of the miRNA expression vector, a gene recombination method usually employed for the target organism species can be used. For example, for plants, the hairpin RNA expression plasmid method can be used. For animals, the siRNA expression vector method can be used. As a system for expressing siRNA, RNA polymeraseasel can be used, and there are a tandem type and a stem loop type. For example, piGENETMhU6 and piGENETMtRNA (iGENE Therapeutics) can be used. Preferably, (i) the siRNA corresponding to the selected miRNA is amplified with primers containing sense and antisense sequences, and the amplified fragment is digested with restriction enzymes.
  • oligonucleotide is inserted downstream of the U6 promoter to make it a tandem type.
  • An oligonucleotide containing sense, loop, and antisense sequences can be annealed and inserted downstream of the U6 promoter (see RNAi Experimental Protocol). "Experimental Medicine Supplement, pages 95-110)
  • the expression vector can be introduced into cells using a cell introduction kit such as EffectinTM.
  • the miRNA expression vector is introduced into a cell or an organism by a well-known method, for example, by electoporation, Ca + polyphosphate method, particle gun method, or the like.
  • an siRNA complementary to the miRNA can be designed.
  • the expression of the protein encoded by the regulated gene can be controlled by the siRNA.
  • the present invention provides a protein encoded by a regulated gene whose expression is controlled by miRNA. It also encompasses the development of treatments for protein-related diseases, and treatments for diseases, using siRNA when the quality is suppressed by the presence of the miRNA.
  • miRNAs can be designed by dividing them into constant regions and variable regions. For example, based on a known miRNA sequence, in the constant region and the variable region, a base sequence having any possibility in the ambiguity set separately is generated, and a miRNA sequence data library is created. The miRNA sequence data thus generated can be searched for the presence of a complementary sequence on the 3 ′ UTR of the mRNA to be subjected to translation control.
  • the gene to be controlled (target gene) controlled by miRNA predicted by the present invention can be used to control gene expression and protein translation by miRNA or siRNA acting on miRNA. Thus, it can be used for the development of a treatment for a disease associated with the protein encoded by the predicted target gene and a treatment for a disease associated with the protein encoded by the predicted target gene.
  • the 5 'side has a common arrangement of several miRNAs, such as let-7a, let-7b, let-7c, let_7d, let_7e, and let_7f, respectively.
  • the motif sequence uGACCUAU exists as a sequence complementary to the above sequence. Based on this tendency, 8 bases on the 5 ′ side of miRNA were defined as “conserved region”, and 9 bases and beyond were defined as “non-conserved region”.
  • a region complementary to the known human miRNA collected in the candidate target base sequence was searched for and used as a complementary region.
  • a search using relatively mild criteria specifically, a region in which two-thirds or more of the base is complementary to the full-length nucleotide sequence of miRNA is selected, and (305).
  • a search based on strict criteria specifically, a base sequence that is continuous at least 8 bases exists. Those were selected and set as target regions (306). As a result, 474 target regions corresponding to 95 human miRNAs were obtained. That is, a gene having a target region for each miRNA is a target gene (controlled gene) (Table 2).
  • fSSOSlT is o— 538SSO "" ⁇ ) 6i-a iuI
  • miR-118 Marauder 144679 From the results obtained in Example 1, three types of miRNAs (miR-17 (SEQ ID NO: 9), miR-29 (SEQ ID NO: 23), and miR-102 (SEQ ID NO: 38)) were selected. One gene was selected from the predicted target genes (NM_001949 (SEQ ID NO: 108), NM020390 (SEQ ID NO: 109), NM_004496 (SEQ ID NO: 110)), and by experiment, each synthetic miRNA was encoded by each target gene. The inhibitory activity on protein translation was examined by observing the effect on the expression of the proteins (E2F3, eIF5A, HNF3alpha) (Fig. 2).
  • DMEM Dulbecco's modified Eagle's Medium
  • FBS fetal bovine serum
  • miR-17, miR_29, and miR-102 shown in Table 1 were respectively synthesized. Each of the above synthetic miRNAs was introduced into human G2 cells at 2 ⁇ ⁇ using 01igofectamin TM (Invitrogen).
  • NM_001949 is an E2F transcription factor 3 [synonym: KIAA0075,
  • E2F3, E2F-3 Transcription factor E2F3, E2F-3].
  • the E2F transcription factor is located at the E2 site recognition site (TTTCC / GCGC) found in the promoter region of the gene encoding a protein involved in cell cycle regulation and gene replication. Binds to DNA in cooperation with the DB protein.
  • the DRTF1 / E2F complex functions in controlling the transition from G1 to S phase of the cell cycle.
  • E2F3 selectively binds to retinoblastoma protein 1 (RB1) depending on the cell cycle. Match. Suppression of the E2F3 protein impairs cell cycle transition, cell division, and differentiation, and correlates with cancer progression and dysregulated E2F2 expression. Therefore, suppression of E2F3 expression by miR-17 can be used to block cell division in human cancer tumors, that is, can be used to treat cancer.
  • Eukaryotic translation initiator factor 5A2 [Synonyms: eIF-5A2 protein, eIF5Ali, eIF_5A2 proteinj.
  • Eukaryotic initiation factor 5A (eIF5A) (eIF-4D, eIF_5A) is a ribosomal peptidyl transferase
  • eIF_5A has been identified as one of the essential cofactors of the HIV-1 transmutabeta protein Rev. Rev plays a key role in the complex regulation of HIV-1 gene expression and therefore in the development of virion infection. Expression of eIF_5A is essential for Rev function, and inhibition of this interaction leads to a block in the viral replication cycle. Therefore, suppression of eIF5A protein expression by miR_29 can be used to block the HIV virus replication cycle, that is, it can be used to treat HIV diseases.
  • NM_004496 is an H-marked atocyte nuclear factor 3, alpha [synonym: HNF3a, HNF3A, MGC33105, TCF3A, forkhead box Al, hepatocyte nuclear factor 3-alpha (HNF-3a) (Forkhead box protein Al)].
  • HNF-3a acts as a transcription activator of many liver genes such as AFP, Albumin, tyrosine aminotransferase, and PEP CK. Interacts with the cis-regulatory regions of these genes.
  • HNF_3a is a member of the forkhead class, a DNA-binding protein. Similar family members in mice have a role in regulating metabolic differences in pancreas and liver.
  • miR_102 suppression of HNF3a protein expression by miR_102 can be used to block the growth of human cancer tumors, that is, it can be used to treat cancer.
  • NMjt ? such as the above numbers (NM_001949, NM.020390, NM_004496) Indicates the gene registration number assigned by BI.
  • expression of a protein encoded by a regulated gene can be controlled by a combination of a miRNA and a regulated gene predicted by the method for searching for a regulated gene of a functional RNA in the present invention. It can be used in the technical field of genetic engineering.
  • FIG. 1 is a flowchart of a miRNA target gene prediction procedure.
  • Fig. 2 shows the combination of miRNA, target gene, and target protein verified by experiments.
  • Figure 3 shows the effect of miRNAs (miR-17, miR-29, miR-102) on the expression of E2F3, eIF5A, and HNF3alpha.
  • Fig. 4 shows examples of motif sequences and miRNAs having the motif sequences.
  • Figure 5 shows examples of miRNAs with the same motif

Abstract

Pour commander l'expression d'un gène par des micro-ARN via le phénomène de la régulation de l'expression génique par ce type d'ARN (type de régulation existant à l'état naturel), on procède à une identification réelle ou présumée de micro-ARN et d'un ou de plusieurs gènes soumis à la régulation visée. Des séquences de base de molécules de micro-ARN sont subdivisées en une région présentant une séquence de base commune à plusieurs micro-ARN (région conservée) et en une autre région présentant des séquences de base communes (région autre que la région conservée, dite région non conservée). On fixe ensuite des ambiguïtés (recherche de niveaux d'exactitude) de degrés différents dans ces régions. En d'autres termes, on fixe une ambiguïté d'un degré relativement lâche dans la région conservée et une ambiguïté de niveau relativement strict dans la région non conservée. A l'aide des séquences de base ainsi obtenus pour la recherche, on recherche pour identification présumée ou réelle un gène présentant une séquence de base complémentaire de la molécule d'ARN fonctionnel (c'est-à-dire un candidat pour le gène soumis à régulation).
PCT/JP2004/011624 2003-08-13 2004-08-12 Methode d'identification reelle ou presumee d'un gene soumis a une regulation commandee par un arn fonctionnel et utilisation de cette methode WO2005017145A1 (fr)

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US8440636B2 (en) 2007-07-31 2013-05-14 The Board Of Regents, The University Of Texas System Micro-RNA family that modulates fibrosis and uses thereof
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