CN103045593A - Micrornas and application thereof - Google Patents

Abstract

Described is a new polynucleotide relative to prostatic cancer and lung cancer. The polynucleotide is an miRNA and miRNA precursor. Disclosed are relative methods and compositions used for diagnosis, prognosis and treatment of these medical condition as well as methods for determining prostatic cancer and lung cancer modifiers.

Description

Microrna and uses thereof

The application is that international filing date is that to enter China, application number be dividing an application of 200580023699.2 the application for a patent for invention that is entitled as " Microrna and uses thereof " for the International Application Serial No. PCT/IB2005/002383 on May 14th, 2005.

Technical field

Present invention relates in general to microRNA molecules and related with it or from its various nucleic acid molecule of deriving.

Background technology

Microrna (miRNA) is the short rna oligonucleotide that participates in about 22 Nucleotide of generegulation.Microrna s suppresses to come regulatory gene to be expressed by target mRNA to cut or to translate.Although miRNAs is present in widely species, comprises and just just it being identified recently among Caenorhabditis elegans (C.elegans), fruit bat (Drosophila) and the people.Heavier is that the effect of miRNA in advancing of disease and progress just begins to obtain understanding as of late.

Because its less size, all the time, the methodology of Application standard is difficult to identify miRNAs.Identified a limited number of miRNA by extracting a large amount of RNA.Also identified the miRNA that can make contributions by the phenotype of visual discrimination to showing.Express array data and show that miRNA expresses in different developmental phases or different tissue.The restriction of miRNA in some tissue or on limited etap shows that the miRNAs that identifies up to now probably is the small portion of total miRNA.

Developed in recent years the computer approach for the identification of remaining miRNA in the genome.Instrument for example MiRscan and MiRseeker had been identified the miRNA that confirmed by experiment afterwards.Based on these PC Tools, estimated that human genome comprises 200-255 miRNA gene.Yet these estimate that based on such hypothesis namely remaining miRNA to be identified has the character identical with the miRNA that has identified.Based on the basic importance of miRNA in mammalian biology and disease, unknown miRNA need to be identified in this area.The present invention has satisfied these needs and miRNA and its purposes of suitable high number is provided.

Summary of the invention

The present invention relates to comprise the nucleic acid of separation of the sequence of pri-miRNA, pre-miRNA, miRNA, miRNA*, anti--miRNA or miRNA binding site or its variant.Described nucleic acid can comprise the sequence of the hairpin structure that relates in the table 1, the sequence of sequence flag symbol (identifiers) 6757248-6894882 of table 10, the sequence flag symbol 1-6318 of table 17 or the sequence of 18728-18960, the sequence of the miRNA that relates in the table 1, the sequence flag symbol 1-117750 of table 10 or the sequence of 6894883-10068177, the sequence flag symbol 6319-18727 of table 17 or the sequence of 18961-19401, the sequence of the target gene binding site that relates in the table 4, the sequence of the sequence flag symbol 117751-6757247 of table 10, its complement, or comprise the sequence of Nucleotide that has at least 12 adjacency of at least 60% identity with it.The nucleic acid of described separation can be 5 to 250 Nucleotide on length.

The present invention also relates to comprise the probe of described nucleic acid.Probe can comprise and table 2 in be called the Nucleotide of at least 8 to 22 adjacency of the miRNA complementation of differential expression in prostate cancer or lung cancer.

The present invention also relates to multiple probe.Described multiple probe can comprise and table 2 in be called at least a probe of each miRNA complementation of differential expression in prostate cancer.Described multiple probe also can comprise and table 2 in be called at least a probe of each miRNA complementation of differential expression in lung cancer.

The present invention also relates to comprise the composition of probe or multiple probe.

The present invention also relates to comprise the biochip of solid substrate, described matrix comprises multiple probe.Each address of determining in the space of described probe is attached on the matrix.Biochip can comprise and table 2 in be called the probe of the miRNA complementation of differential expression in prostate cancer.Biochip also can comprise and table 2 in be called the probe of the miRNA complementation of differential expression in lung cancer.

The present invention also relates to detect the method for differential expression of the miRNA of disease association.Biological sample can be provided and measure with following sequence to have the level of the nucleic acid of at least 70% identity, described sequence is the sequence of the miRNA that relates in the table 1, sequence flag symbol 1-117750 or the sequence of 6894883-10068177, the sequence flag symbol 6319-18727 of table 17 or sequence or its variant of 18961-19401 of table 10.Compare the difference indication differential expression on the nucleic acid level with contrast.

The present invention also relates to identify the method for the compound of regulating pathological conditions.Can provide and can express the cell that has the nucleic acid of at least 70% identity with following sequence, described sequence is the sequence of the miRNA that relates in the table 1, sequence flag symbol 1-117750 or the sequence of 6894883-10068177, the sequence flag symbol 6319-18727 of table 17 or sequence or its variant of 18961-19401 of table 10.Described cell can be contacted with candidate modulator, then measure the expression level of described nucleic acid.Compare with contrast, the difference on the nucleic acid level identifies that this compound is the conditioning agent of the pathological conditions related with this nucleic acid.

The present invention also relates to suppress target gene in the method for cells.Can in cell, import nucleic acid with the amount that is enough to suppress expression of target gene.Target gene can comprise basically sequence or its variant of the sequence flag symbol 117751-6757247 of the binding site identical with the binding site that relates in the table 4, table 10.Nucleic acid can wrap the sequence of SEQ ID NOS:1-760616, the sequence flag symbol 1-117750 of table 10 and sequence, the sequence shown in the table 17 or its variant of 6757248-10068177.Can in external or body, suppress the expression of target gene.

The present invention also relates in cell, increase the method for the expression of target gene.Can in cell, import nucleic acid with the amount that is enough to increase expression of target gene.Target gene can comprise basically sequence or its variant of the sequence flag symbol 117751-6757247 of the binding site identical with the binding site that relates in the table 4, table 10.Nucleic acid can comprise and sequence, the sequence shown in the table 17 or its variant of the sequence flag of SEQ ID NOS:1-760616, table 10 symbol 1-117750 and 6757248-10068177 complementary sequence basically.Can in external or body, suppress the expression of target gene.Can in external or body, increase the expression of target gene.

The present invention also relates to treat the patient's who suffers from the illness shown in the table 6 method, it comprises to its patient of needs uses such nucleic acid, and described nucleic acid comprises sequence or its variant shown in the sequence, the sequence shown in the table 10, table 17 of SEQ ID NOS:1-760616.

Description of drawings

Fig. 1 shows the model of the maturing of miRNA.

Fig. 2 shows the cluster signal explanation of (cluster) of the MC19 on the 19q13.42.Little figure A show No. 19 chromosomal about 500, the zone of 000bp, from 58,580,001 to 59,080,000 (according to the assembling of in May, 2004 USCS), wherein this present position that clusters comprises the gene of adjacent proteins encoded.Represent that with rectangle MC19-1 clusters.Represent Mir-371, mir-372 and mir-373 with line.Represent that with large arrow flank connects the gene of this proteins encoded that clusters.Little figure B shows the detailed construction that MC19-1miRNA clusters.Shown approximately～102, the zone of 000bp, from 58,860,001 to 58,962,000 (according to the assembling of in May, 2004 USCS).Represent the miRNA precursor with black bar.It should be noted that all miRNA are in equidirectional from left to right.Shadow region around the miRNA precursor represents the wherein repeating unit of embedded precursor.The position that has also shown mir-371, mir-372 and mir-373.

Fig. 3 is the graphic representation that the multiple sequence of 35 people repeating units (A) on the different sizes of about 690nt and 26 chimpanzee repeating units (B) is compared.This chart is that the similarity scoring that everybody puts by calculating in the comparison of the moving window (sliding window) that uses average 10nt (maximum scores-1, minimum scoring-0) produces.Compare repeating unit's sequence by the ClustalW program.For giving the scoring that is illustrated in this locational similarity degree in each position of gained comparison.Demarcate with vertical line in the zone that comprises the miRNA precursor.Indicate from the definite position of the ripe miRNA of the 5 ' stem (5p) of precursor and 3 ' stem (3p) generation with vertical line.

Fig. 4 shows the sequence alignment of 43 A type pre-miRNA that MC19-1 clusters.Little figure A shows the multiple sequence comparison of using collimation mark to be shown as ripe miRNA position.The bottom shows consensus sequence.Following with the conservative Nucleotide of color marking: black 100%, Dark grey 80% to 99% and light gray (clear grey) 60% to 79%.The sequence alignment that the upper visitor of the total ripe A type miRNA of little figure B demonstration and mir-371, mir-372, miR-373 clusters.Little figure C shows the comparison that total ripe A type miRNA and hsa-mir-371-373 mouse directly cluster to homology.

Fig. 5 shows the expression analysis of MC19-1miRNA.Little figure A shows the rna blot analysis of the A type miRNA of 2 selections.Use is analyzed expression from total RNA of human brain (B), liver (L), thymus gland (T), placenta (P) and HeLa cell (H).The ethidium bromide staining of the expression of mir-98 and tRNA band is with comparing.Little figure B shows that the RT-PCR of the mRNA transcript that comprises A type miRNA precursor analyzes.Use few deoxythymidine to carry out

Reverse transcription from total RNA of placenta.Use afterwards the primer (being indicated by the horizontal arrow mark) of appointment to carry out PCR.The zone that checks is illustrated up.Vertical black bar represents pre-miRNA; Shadow region around pre-miRNA represents repeating unit; Indicate the position of 4 EST on the right side; Indicate polyadenylic acid (poly-A) site with perpendicular arrow mark, such as the site of in EST, finding and be positioned at AATAAA consensus sequence downstream.Below the district's explanation that clusters, show the fragment of using 3 combination of primers expections to obtain from RT-PCR.Below the fragment of expection, shown the result that RT-PCR analyzes.Little figure C shows the sequencing strategy of FR2 fragment.With this fragment be cloned into pTZ57R T carrier and use outside (external) and inside (internal) primer checks order.

Embodiment

The invention provides the nucleotide sequence of miRNA, its precursor, its target and correlated series.These nucleic acid are used for diagnostic purpose, and also are used for the modified target dna expression.By following description of the invention, it is very clear that other aspects of the present invention will become to those skilled in the art.

1. definition

Before this compound of disclosure and description, product and composition and method, be appreciated that terminology used herein is to be used for describing particular, rather than be used for limiting.Must be pointed out, as used in specification sheets and the appended claim, singulative " one (a) ", " one (an) " and " should (the) ", unless context clearly point out in addition, comprise plural indicator (plural referent).

A. animal

" animal " used herein can refer to fish, Amphibians, Reptilia, bird and Mammals, for example mouse, rat, rabbit, goat, cat, dog, cow, ape and people.

B. adhere to

" adhere to " as used herein or " fixing ", when referring to probe and solid support, can represent between probe and the solid support be combined in conjunction with, wash, analyze and remove under the condition of (removal) and be enough to keep stable.In conjunction with can be covalency or non-covalent.Can be between probe and solid support directly form covalent linkage, maybe can or be included in specific reactive group on solid support or the probe or be included on two molecules by linking agent and form covalent linkage.Non-covalent combination can be one or more in static, the hydrophilic and hydrophobic interaction.Non-covalent combination comprises for example non-covalent combination of streptavidin to the covalent attachment of upholder and biotinylated probe to streptavidin of molecule.Immobilization also comprises the combination of covalency and noncovalent interaction.

C. biological sample

" biological sample " used herein can refer to comprise biological organization or the fluid sample of nucleic acid.These samples include, but not limited to from the tissue of animal separation.Biological sample also can comprise the section of tissue, for example the freezing microtome section, blood, blood plasma, serum, phlegm, ight soil, tears, mucus, hair and the skin that are used for the histology purpose of examination of living tissue and necrotomy sample, collection.Biological sample also comprises explant and from the former generation of patient tissue and/or the cell culture of conversion.Can provide biological sample by taking out cell sample from animal, but the cell that separates before also can passing through to use (for example, separated in another by another person time, and/or be used for another kind of purpose) or obtain biological sample by carrying out in vivo method of the present invention.Also can use archives economy (archival tissue), for example have the tissue for the treatment of or outcome history.

D. complementary

" complementation " used herein or " complementation " can refer to Nucleotide or the Watson-Crick between the nucleotide analog or the Hoogsteen base pairing of nucleic acid molecule.

E. differential expression

" differential expression " can phalangeal cell and tissue within and between qualitative or quantitative difference on time and/or cellular gene expression pattern.Therefore, the gene of differential expression can make its expression be changed qualitatively, for example is included in healthy tissues to the activation in the diseased tissue or inactivation.With respect to another kind of state, gene can open or close under particular state, thereby allows the comparison of two or more states.Be subjected to the gene of qualitative adjusting in state or cell type, to represent the expression pattern that can detect by standard technique.Some genes will be expressed in a kind of state or cell type, but all not express in both cases.Selectively, the difference in the expression is quantitative for example regulate expressing this point, describedly is adjusted to rise, thereby causes the amount of the transcript that increases, perhaps downward modulation, thus cause the amount of the transcript that reduces.Expressing different degree only need to even as big as the characterization technique by standard, for example express array (expression arrays), quantitative reverse transcriptase PCR, rna blot analysis and RNA enzyme protection and carry out quantitatively getting final product.

F. gene

" gene " used herein can be to comprise to transcribe and/or translate the genomic gene of regulating sequence and/or coding region and/or non-translated sequence (for example, intron, 5 '-and 3 '-non-translated sequence).The coding region of gene can be the nucleotide sequence of encoding amino acid sequence or functional r NA (for example tRNA, rRNA, catalytic RNA, siRNA, miRNA and sense-rna).Gene also can be corresponding to coding region (for example, exon and miRNA), randomly comprise and be connected to 5 ' on it-or mRNA or the cDNA of 3 '-non-translated sequence.Gene also can be the modified nucleic acid molecule in external generation, and this nucleic acid molecule comprises all or part coding region and/or is connected to 5 ' on it-or 3 '-non-translated sequence.

G. host cell

" host cell " used herein can be the cell of natural generation or comprise carrier and the cell of the conversion that the described carrier of support copies.Host cell can be cultured cells, explant, cells in vivo etc.Host cell can be for example intestinal bacteria (E.coli) of prokaryotic cell prokaryocyte, or eukaryotic cell, for example yeast, insect, Amphibians or mammalian cell, for example CHO, HeLa.

H. identity

This is in identical Nucleotide or amino acid whose sequence that " same " used in the context of two or more nucleic acid or peptide sequence or " identity " can refer to have in the specific region particular percentile.Calculate by the following method per-cent, two sequences of relatively more optimum comparison, compare two sequences in the specific region, determine on two sequences, all to occur the number of position of identical residue with the number of the position that produces coupling, with the coupling the position number divided by position number total in the specific region, and acquired results be multiply by 100, to produce the per-cent of sequence identity.Different or comparison produces staggered end and specific region relatively only has in the situation of unique sequence at two sequence lengths, the residue of unique sequence is included in the denominator of calculating rather than in the molecule.When comparison dna and RNA, thymus pyrimidine (T) and uridylic (U) are considered to be equal to.But artificially or by for example BLAST or BLAST 2.0 calculate identity with the computer sequence algorithm.

I. mark

" mark " used herein can refer to pass through the composition that spectrum, photochemistry, biological chemistry, immunochemistry, chemistry or other physics means detect.For example, useful mark comprises ³²P, fluorescence dye, the close reagent of electronics, enzyme (for example, the enzyme as in ELISA, generally using), vitamin H, digoxigenin or haptens and other entities that can be detected.Can mark be integrated into nucleic acid and albumen in any position.

J. nucleic acid

" nucleic acid " used herein or " oligonucleotide " or " polynucleotide " can refer at least two covalently bound Nucleotide together.As skilled in the art to understand, the sequence of complementary strand has also been determined in the description of strand.Therefore, nucleic acid also comprises the complementary strand of the strand that is described.Also as skilled in the art to understand, many variants of nucleic acid can with given nucleic acid the same be used for identical purpose.Therefore, nucleic acid also comprises nucleic acid and the complement that it is substantially the same.As recognized by those skilled in the art, strand provides the probe that can hybridize with target sequence under stringent hybridization condition.Therefore, nucleic acid is also contained in the probe of hybridizing under the stringent hybridization condition.

Nucleic acid can be strand or double-stranded, maybe can comprise the part of double-stranded and single stranded sequence.Nucleic acid can be DNA, be genomic dna and cDNA, RNA or hybrid, its amplifying nucleic acid can comprise the combination of deoxyribonucleotide and ribonucleotide, and the combination of base, described base comprises uridylic, VITAMIN B4, thymus pyrimidine, cytosine(Cyt), guanine, Trophicardyl, xanthine xanthoglobulin, iso-cytosine (isocytosine) and isoguanine.Can obtain nucleic acid by the method for chemosynthesis or by recombination method.

Nucleic acid generally comprises phosphodiester bond, although can comprise such nucleic acid analog, this analogue can have at least one different key, for example, and phosphoramidate, thiophosphatephosphorothioate, phosphorodithioate or O-methyl phosphoramidite key and peptide nucleic acid(PNA) main chain and key.Other analogue nucleic acid comprise have positive main chain, nonionic main chain and non-ribose main chain those, comprise U.S. Patent number 5,235, analogue nucleic acid of describing in 033 and 5,034,506 (it is incorporated herein by reference).A definition of nucleic acid also comprises the nucleic acid that comprises one or more non-naturals Nucleotide that occur or modified.Modified nucleotide analog also can be positioned at for example 5 '-terminal and/or 3 '-end of nucleic acid molecule.The representative example of nucleotide analog can be selected from sugar or the modified ribonucleotide of main chain.Yet, should be understood that, the modified ribonucleotide of nuclear base (nucleobase), be that such ribonucleotide also is suitable, this ribonucleotide comprises non-natural nuclear base that occur rather than natural generation, for example modified uridine or cytidine on 5 positions, for example 5-(2-is amino) propyl group uridine, 5-broxuridine; Modified adenosine and guanosine on 8 positions, for example 8-bromine guanosine; Denitrogenation Nucleotide, for example 7-denitrogenation-adenosine; O-and N-alkylation Nucleotide, for example N6-methyladenosine.2 '-the OH base can be selected from H, OR, R, halogen, SH, SR, NH ₂, NHR, NR ₂Or the replacement of the group of CN, wherein R is C ₁-C ₆Alkyl, alkenyl or alkynyl, and halogen is F, Cl, Br or I.Can carry out because of a variety of causes the modification of ribose phosphate backbone, for example for the stability that in physiological environment, increases these molecules and transformation period or for as the probe on the biochip.Can prepare the nucleic acid of natural generation and the mixture of analogue; Mixture and the nucleic acid of natural generation and the mixture of analogue that selectively, can prepare the different IPs acid-like substance.

K. be operably connected

Under the spatially control of connected promotor of the expression that " being operably connected " used herein can refer to gene.Promotor can be located at 5 ' (upstream) or 3 ' (downstream) of the gene under its control.Distance between promotor and the gene can and the gene of this promotor and its control in the gene in this promotor source between distance roughly the same.As known in the art, can regulate the variation in this distance and do not lose the function of promotor.

L. probe

" probe " used herein can refer to such oligonucleotide, and this oligonucleotide can be by the chemical bond of one or more types, usually by the complementary base pairing, usually by the next target nucleic acid in conjunction with complementary sequence of the formation of hydrogen bond.Depend on the stringency of hybridization conditions, probe can be in conjunction with the target sequence of incomplete and this probe sequence complementation.The base-pair mismatch that can have any number, this base-pair mismatch is disturbed the hybridization between target sequence and the single-chain nucleic acid of the present invention.Yet if can not hybridize under the minimum stringency of hybridization conditions even the number of sudden change is large like this, so described sequence is not complementary target sequence.Probe can be strand or part strand and partially double stranded.The structure of target sequence, composition and character determine the chain (strandedness) of probe.Directly label probe or indirect labelling probe are for example used the biotinylated probe of subsequently combination of streptavidin mixture.

M. promotor

" promotor " used herein can refer to can give, activate or strengthen the synthetic or natural molecule of deriving of expression of nucleic acid in cell.Promotor can comprise one or more further enhancings expression and/or change the space expression of homologous genes and/or the specificity regulatory element of time expression.Promotor also can comprise far-end enhanser or repressor element, and it can be positioned at from the initiation site of transcribing and reach the right position of several kilobase.Promotor can derive from such source, and this source comprises virus, bacterium, fungi, plant, insect and animal.But the expression of promotor composing type ground regulatory gene component, or cell, tissue or organ about occuring therein to express, or the etap about occuring therein to express, or at the response external stimulus, for example expression of difference ground regulatory gene component in physiological stress, pathogenic agent, metal ion or the inductor.The representative example of promotor comprises phage t7 promotor, phage T3 promotor, SP6 promotor, lac operator gene-promotor, tac promotor, SV40 late promoter, SV40 early promoter, RSV-LTR promotor, CMV IE promotor, SV40 early promoter or SV40 late promoter and CMV IE promotor.

N. selective marker

" selective marker " used herein can refer to any such gene, and this gene is that such cell is given phenotype, in this cell, expresses this gene to help to identify and/or select to use the cell of gene construct transfection or conversion.The representative example of selective marker comprises ampicillin resistance gene (Amp ^r), tetracycline resistance gene (Tc ^r), bacterium kalamycin resistance gene (Kan ^r), zeocin resistant gene, gene and the luciferase genes of giving AURI-C gene to the resistance of microbiotic aureobasidin A, phosphinothricin resistant gene, neomycin phosphotransferase gene (nptII), hygromycin gene, β-glucuronidase (GUS) gene, E.C. 2.3.1.28 (CAT) gene, encoding green fluorescent protein.

O. stringent hybridization condition

" stringent hybridization condition " used herein can refer to such condition, under this condition, in the complex mixture of for example nucleic acid, the first nucleotide sequence (for example, probe) can hybridize with the second nucleotide sequence (for example, target), but other sequence hybridizations of getting along well.Stringent condition is sequence dependent, and is different in different environment.Usually, select stringent condition, make it than the pyrolysis chain temperature (T of the particular sequence under the ionic strength pH that determines _m) low about 5-10 ℃.T _mCan be such temperature (under ionic strength, pH and the nucleic acid concentration determined), under this temperature, 50% and the probe target complementation in equilibrium state and target sequence hybridization (because the excessive existence of target sequence, so at T _mLower, 50% probe is occupied in equilibrium state).Stringent condition can be such condition, in this condition, at pH7.0 to 8.3 time, salt concn is lower than about 1.0M sodium ion, be typically about 0.01-1.0M Na ion concentration (or other salt), and be about at least 30 ℃ for short probe (for example, about 10 to 50 Nucleotide) temperature, be about at least 60 ℃ for long probe (for example, more than about 50 Nucleotide) temperature.Also can by add destabilizing agent for example methane amide obtain stringent condition.For selectivity or specific hybrid, positive signal can be at least 2 to 10 times of background hybridization.Exemplary stringent hybridization condition comprises following: 50% methane amide, 5xSSC and 1%SDS, and at 42 ℃ of lower incubations, or 5xSSC, 1%SDS, at 65 ℃ of lower incubations, in 0.2x SSC and 0.1%SDS, washing under 65 ℃.

P. basically complementary

Used herein " basically complementary " can refer to 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50 or the zone of more Nucleotide on, the complement of First ray and the second sequence has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, or two sequences are hybridized under stringent hybridization condition.

Q. basically same

" basically same " used herein can refer to 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50 or more Nucleotide or amino acid whose zone on, First ray and the second sequence have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, perhaps for nucleic acid, if the complement of First ray and the second sequence is basically complementary.

R. target

" target " used herein can refer to can be by the polynucleotide of one or more probe combinations under stringent hybridization condition.

S. terminator

" terminator " used herein can refer to the sequence that is positioned at transcription unit's end, the signal of Transcription Termination is provided.Terminator can be to comprise 3 ' of polyadenylation signal-non-translation DNA sequence, and this sequence can help the polyadenylic acid sequence to add to 3 ' of primary transcript-end.Terminator can be from such source, and this source comprises virus, bacterium, fungi, plant, insect and animal.The representative example of terminator comprises the SV40 polyadenylation signal, HSV TK polyadenylation signal, the CYC1 terminator, the ADH terminator, the SPA terminator, nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens (Agrobacterium tumefacien), the terminator of cauliflower mosaic virus (CaMV) 35S gene, zein spirit-soluble gene terminator from Zea mays (Zea mays), the gene terminator sequence of the small ylidene gene of rubisco (SSU), the terminator of subclover stunt virus (SCSV) gene order, do not rely on intestinal bacteria terminator and the lacZ α terminator of ρ.

T. carrier

" carrier " used herein can refer to comprise the nucleotide sequence of replication origin.Carrier can be plasmid, phage, bacterial artificial chromosome or yeast artificial chromosome.Carrier can be DNA or RNA carrier.Carrier can be the outer carrier of the karyomit(e) of self-replacation or the carrier that is integrated into host genome.

2. Microrna

Not bound by theory, Fig. 1 shows the present model of the maturing of Mammals miRNA.The gene of transcribed coding miRNA, thus cause being called the generation of the miRNA precursor of pri-miRNA.Pri-miRNA can be the part that comprises the polycistron RNA of a plurality of pri-miRNA.Pri-miRNA can form the hairpin structure with stem and ring.As shown in fig. 1, stem can comprise the base of mispairing.

The hairpin structure of pri-miRNA can be identified by Drosha, and it is RNA enzyme III endonuclease.Drosha can identify the end-rings of pri-miRNA and roughly cut two spiral corners in stem, to produce the precursor that is called pre-miRNA of 60-70nt.Drosha can cut pri-miRNA, has the typical stagger of RNA enzyme III endonuclease, has the pre-miRNA stem ring of 3 ' overhang of 5 ' phosphoric acid and about 2 Nucleotide with generation.An about spiral corner (about 10 Nucleotide) that extends beyond the stem of Drosha cleavage site may be that effectively processing is necessary.Then can be with pre-miRNA from the nucleus active transport to kytoplasm by Ran-GTP and output acceptor (export receptor) Ex-portin-5.

Dicer also is RNA enzyme III endonuclease, can identify pre-miRNA.Dicer can identify the double-stranded stem of pre-miRNA.Dicer also can identify 5 ' phosphoric acid and the 3 ' overhang of stem cyclic group section.Dicer can excise end-rings in two spiral nooks of distance stem cyclic group section, thereby stays 3 ' overhang of 5 other ' phosphoric acid and about 2 Nucleotide.The siRNA sample duplex of the comprised mispairing of gained comprises ripe miRNA and the fragment of the similar size that is called miRNA*.MiRNA and miRNA* can derive from the opposite arm of pri-miRNA and pre-miRNA.Can in the library of the miRNA that clones, find the miRNA* sequence, but its frequency generally is lower than miRNA.

Although occur with the double-stranded kind with miRNA* at first, miRNA finally can be integrated into the form of single stranded RNA the ribonucleoprotein complex that is called as the reticent mixture (RISC) that RNA induces.Various albumen can form RISC, and this can cause being loaded into RISC for which the bar chain in the binding site of the specificity of miRNA/miRNA* duplex, target gene, the activity of miRNA (suppressing or activation), the miRNA/miRNA* duplex and change.

When the miRNA chain with the miRNA:miRNA* duplex was loaded among the RISC, miRNA* can remove and degrade.The chain that is loaded into the miRNA:miRNA* duplex among the RISC can be its 5 ' terminal chain that does not have tight pairing.Two ends at miRNA:miRNA* all have in the situation of 5 ' pairing about equally, and miRNA and miRNA* can have the activity of silencer.

RISC can based on the high-caliber complementarity between miRNA and the mRNA, particularly by the Nucleotide 2-8 of miRNA, identify target nucleic acid.Only have an example to report in animal, in this example, the interaction between miRNA and its target is along the whole length of miRNA.In mir-196 and Hox B8, show this situation, and also shown cutting people 2004 such as (, Science 304-594) Yekta of mir-196 mediation Hox B8 mRNA.In other respects, known these interact and only are present in (Bartel ﹠amp in the plant; Bartel 2003, Plant Physiol 132-709).

Many researchs have been investigated as obtaining enough translations and have been suppressed, and the base pairing between miRNA and its mRNA target requires (Bartel 2004, the summary of Cell 116-281).In mammalian cell, front 8 Nucleotide of miRNA may be important (Doench ﹠amp; Sharp 2004GenesDev 2004-504).Yet other parts of Microrna also can participate in the mRNA combination.In addition, 3 ' the sufficient base pairing located can compensate at 5 ' insufficient pairing of locating (people such as Brennecke, 2005 PLoS 3-e85).5 ' the base 2-7 that has pointed out miRNA in the Calculation and Study that whole genome is analyzed the miRNA combination target in conjunction with in special role, but also admit usually to be found to be effect people 2005 Cell120-15 such as () Lewis of the 1st Nucleotide of " A ".Similarly, the people such as Krek (2005, Nat Genet 37-495) use Nucleotide 1-7 or 2-8 to identify and the checking target.

Target site among the mRNA can be at 5 ' UTR, 3 ' UTR or in the coding region.Enjoyably, multiple miRNA can be by identifying identical or identical mRNA target is regulated in a plurality of sites.In the target of most Genetic identification, exist the complementary site of a plurality of miRNA can show that the synergy of a plurality of RISC provides the most effective translation restraining effect.

Any mechanism during miRNA can suppress by two kinds of mechanism: mRNA cuttings or translation instructs the RISC down-regulation of gene expression.If mRNA and miRNA have complementarity to a certain degree, this miRNA can cut this mRNA specifically so.When miRNA instructs cutting, otch can and the Nucleotide of the residue 10 of miRNA and 11 pairings between.Selectively, if miRNA does not have desired degree and complementarity miRNA, miRNA can suppress translation so.Translation is suppressed at may be more general in the animal, because animal can have the more complementarity of low degree.

Should be pointed out that in 5 of a pair of miRNA in office and miRNA* ' and the 3 ' end and can have mutability.This mutability may be because the mutability that exists in Drosha and the Dicer enzymatic processing for cleavage site causes.The terminal mutability of 5 of miRNA and miRNA* ' and 3 ' also can be because the mispairing in the stem structure of pri-miRNA and pre-miRNA causes.The mispairing of stem chain can cause many different hairpin structures.Mutability in the product that mutability in the stem structure also can cause producing by Drosha and Dicer cutting.

3. nucleic acid

The nucleic acid that the present invention relates to separate, the nucleic acid of described separation comprise sequence or its variant shown in nucleotide sequence, the sequence shown in the table 10 and the table 17 that relates among the SEQ ID NOS:1-760616.Variant can be the complement of the nucleotide sequence of reference.Variant also can be basically same with reference nucleotide sequence or its complement nucleotide sequence.Variant also can be under stringent condition and with reference to nucleotide sequence, its complement or basically with the nucleotide sequence of its same nucleotide sequence hybridization.

Nucleic acid can have the length of 10 to 100 Nucleotide.Nucleic acid can have the length of at least 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,60,70,80 or 90 Nucleotide.But synthetic gene (in external or body) express nucleic acid in cell that nucleic acid or use the following describes.Can be with the form nucleic acid of single chain molecule and with it with complementary nucleic acid hybridization is to form duplex basically, this duplex is taken as nucleic acid of the present invention.Can be with strand or double-stranded form with nucleic acid transfered cell, tissue or organ, maybe can be expressed by synthetic gene by using method well known to those skilled in the art, described method comprises U.S. Patent number 6,506, the method described in 559 (being incorporated herein by reference).

a.Pri-miRNA

Nucleic acid of the present invention can comprise the sequence of pri-miRNA or its variant.The pri-miRNA sequence can comprise 45 to 250,55 to 200,70 to 150 or 80 to 100 Nucleotide.Pre-miRNA, miRNA and miRNA* shown in below the sequence of pri-miRNA can comprise.Pri-miRNA also can comprise miRNA or miRNA* and its complement with and variant.Pri-miRNA can comprise the acid of at least 19% adenosine nucleoside, at least 16% cytidylic acid(CMP), at least 23% thymidylic acid and at least 19% guanylic acid.

Pri-miRNA can form hairpin structure.Hairpin structure can comprise the first and second basically complementary nucleotide sequences.The first and second nucleotide sequences can have 37 to 50 Nucleotide.Can separate the first and second nucleotide sequences by the 3rd sequence of 8 to 12 Nucleotide.As by using the people such as Hofacker, Vienna algorithm described in the Monatshefte f.Chemie 125:167-188 (1994) (its content is incorporated herein by reference), utilize default parameter to calculate, hairpin structure can have the free energy that is less than-25 kcal/mol.Hairpin structure can comprise the end-rings of 4-20,8-12 or 10 Nucleotide.

The sequence of pri-miRNA can comprise the sequence of the hairpin structure that relates in the table 1, the sequence flag of table 10 accords with the sequence flag symbol 1-6318 of the sequence of 6757248-6894882, table 17 or sequence or its variant of 18728-18960.

b.Pre-miRNA

Nucleic acid of the present invention also can comprise the sequence of pre-miRNA or its variant.The pre-miRNA sequence can comprise 45 to 90,60 to 80 or 60 to 70 Nucleotide.MiRNA and miRNA* shown in below the sequence of pre-miRNA can comprise.Pre-miRNA also can comprise miRNA or miRNA* and its complement with and variant.The sequence of pre-miRNA also can be the sequence of pri-miRNA of removing 5 ' and 3 ' terminal 0-160 the Nucleotide of pri-miRNA.

The sequence of pre-miRNA can comprise the sequence of the hair clip that relates in the table 1, the sequence flag of table 10 accords with the sequence flag symbol 1-6318 of the sequence of 6757248-6894882, table 17 or sequence or its variant of 18728-18960.

c.miRNA

Nucleic acid of the present invention also can comprise the sequence of miRNA, miRNA* or its variant.MiRNA sequence can comprise 13 to 33,18 to 24 or 21 to 23 Nucleotide.The sequence of miRNA can be front 13 to 33 Nucleotide of pre-miRNA.The sequence of miRNA can be last 13 to 33 Nucleotide of pre-miRNA.

The sequence of miRNA can comprise the sequence of the miRNA that relates in the table 1, sequence flag symbol 1-117750 or the sequence of 6894883-10068177, the sequence flag symbol 6319-18727 of table 17 or sequence or its variant of 18961-19401 of table 10.

D. resist-miRNA

Nucleic acid of the present invention also can comprise the activity that can block miRNA or miRNA* anti--sequence of miRNA.Anti--miRNA can comprise altogether 5 to 100 or 10 to 60 Nucleotide.Anti--miRNA also can comprise altogether at least 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 or 26 Nucleotide.The sequence of anti--miRNA can comprise (a) basically with 5 ' same at least 5 Nucleotide of miRNA and basically with at least 5-12 Nucleotide of the flanking region complementation of 5 ' of described miRNA terminal target site, or (b) basically with 3 ' same at least 5 to 12 Nucleotide of miRNA and basically with at least 5 Nucleotide of the flanking region complementation of the target site of the 3 ' end of described miRNA.

Anti--sequence of miRNA can comprise the sequence of the miRNA that relates in the table 1, the sequence flag symbol 1-117750 of table 10 or the sequence of 6894883-10068177, sequence flag symbol 6319-18727 or the sequence of 18961-19401 or the complement of its variant of table 17.

E. the binding site of target

Nucleic acid of the present invention also can comprise the sequence of target miRNA binding site or its variant.The target site sequence can comprise altogether 5 to 100 or 10 to 60 Nucleotide.The target site sequence can comprise the sequence of the target gene binding site that relates in the table 4, the sequence flag of table 10 accords with the sequence of 117751-6757247 or at least 5 Nucleotide of its variant.

4. the gene that synthesizes

The present invention also relates to comprise the synthetic gene that may be operably coupled to the nucleic acid of the present invention of transcribing and/or translate the adjusting sequence.The expression that described synthetic gene can be modified the target gene of the binding site with nucleic acid of the present invention.Can be in cell, tissue or organ the expression of modified target dna.Gene that can be synthetic or by the recombinant technology of the standard synthetic gene of from the gene of natural generation, deriving.Synthetic gene also can comprise terminator at 3 '-end of the transcription unit of synthetic gene order.Synthetic gene also can comprise selective marker.

5. carrier

The present invention also relates to comprise the carrier of synthetic gene of the present invention.Carrier can be expression vector.Expression vector can comprise other element.For example, expression vector can have two kinds of such dubbing systems, and described dubbing system can make it be maintained in two kinds of biologies, for example at the Mammals of be used for expressing or insect cell be used for the clone and the prokaryotic cell prokaryocyte of amplification.For integrating expression vector, expression vector can comprise the sequence of at least one and host cell gene group homology, and is connected two flanks and connects the homologous sequence of this expression construct.Can be with the specific gene seat of conformability carrier guiding host cell by selecting suitable homologous sequence to be used for to include carrier.Carrier also can comprise the selectable marker gene that makes it possible to select the host cell that transforms.

6. host cell

The present invention also relates to comprise the host cell of carrier of the present invention.Described cell can be bacterium, fungi, plant, insect or zooblast.

7. probe

The present invention also relates to comprise the probe of nucleic acid of the present invention.Probe can be used for screening and diagnostic method, such as the following method of summarizing.Probe can be attached to or be fixed on the solid substrate biological example chip.

Probe can have the length of 8 to 500,10 to 100 or 20 to 60 Nucleotide.Probe also can have the length of at least 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,60,70,80,90,100,120,140,160,180,200,220,240,260,280 or 300 Nucleotide.Probe also can comprise the joint sequence of 10 to 60 Nucleotide.

8. biochip

The present invention also relates to biochip.Biochip can comprise such solid substrate, and described solid substrate comprises probe of the present invention or a plurality of probe that adheres to.Described probe may be hybridized with target sequence under stringent hybridization condition.The address that can spatially determine is attached to matrix with probe.Can use each target sequence to surpass 1 probe, wherein use overlapping probe or for the probe of the different piece of particular target sequence.Probe may with the hybridization of the related target sequence of single illness.

Can many methods, method as skilled in the art to understand is attached to biochip with probe.Can first synthesising probing needle, then it is attached to biochip, or direct synthesising probing needle on biochip.

Solid substrate can be such material, and this material can be modified to comprise is suitable for adhering to or the Single locus of the separation of combination of probe, and can process by at least a detection method.The representative example of matrix comprises glass, plastics (comprising multipolymer, polypropylene, polyethylene, polybutene, urethane, TeflonJ of acrylics, polystyrene and vinylbenzene and other materials etc.), polysaccharide, nylon or nitrocellulose, resin, the silica of glass and modification or functionalization or based on material (comprising silicon and modified silicon), carbon, metal, unorganic glass and the plastics of silica.Described matrix can allow optical detection and not send out any fluorescence.

Matrix can be the plane, although also can use the matrix of other configurations.For example, for circulation sample analysis (flow-through sample analysis), probe can be placed the inner surface of pipe so that sample volume is minimized.Similarly, matrix can be flexible, and for example flexible foam comprises the closed cell foam of being made by special plastics.

Available chemical functional group's derivatize biochip and probe are to be used for both adhere to subsequently.For example, available chemical functional group (including, but not limited to amino, carboxyl, oxo group or thiol group) derivatize biochip.Use these functional groups, can use directly or indirectly joint, utilize the functional group on the probe to adhere to described probe.Can probe be attached to solid support by 5 ' end, 3 ' end or by the inner core thuja acid.

Also can probe be attached to solid support non-covalently.For example, can prepare biotinylated oligonucleotide, it can in conjunction with the surface with the streptavidin covalently coating, adhere to thereby produce.Selectively, but operation technique for example photopolymerization and photolithography at surperficial synthesising probing needle.

9.miRNA expression analysis

The present invention also relates to identify the method for the miRNA related with disease or pathological condition, it comprises the amount that biological sample and probe of the present invention or biochip is contacted and detect hybridization.Can use the nucleic acid in the pcr amplification sample, this can provide higher sensitivity.

The ability of identifying such miRNA can provide high resolving power, highly sensitive data set (datasets), described miRNA, compare with contrast, overexpression or expression deficiency in pathological cells, described data set can be used for diagnosis, treatment, drug development, pharmacogenetics, biosensor development field and other association areas.The expression overview that is produced by present method can be " finger printing " about the state of the sample of many miRNA.Although two states can have any specific miRNA of similar expression, estimate simultaneously the genetic expression overview that many miRNA make it possible to produce the state of characterize cells.That is, healthy tissues and ill tissue division can be opened.The expression overview of the tissue by relatively being in known various disease state can obtain about each the related information in which kind of miRNA and these states.Thereby, can carry out or confirm that diagnosis is to determine whether tissue sample has the expression overview of normal or diseased tissue.This can provide conditions associated molecular diagnosis.

10. determine expression level

The present invention also relates to determine the method for the expression level of illness cognation miRNA, it comprises biological sample contact and measure the amount of hybridizing with probe of the present invention or biochip.The expression level of disease association miRNA is the information of various ways.For example, can be with the differential expression of the disease association miRNA that compares with contrast with the diagnosis of making the patient is suffered from this disease.The expression level of illness cognation miRNA also can be used for treatment and the morbid state of monitored patient.In addition, the expression level of disease association miRNA can allow to screen for changing the specific drug candidates of expressing the expression overview of overview or inhibition and disease association.

Sample that can be by will comprising target nucleic acid and biochip (this biochip comprise adhere to fully and the probe of target nucleic acid complementation) contact and detect to be higher than and control level detect this target nucleic acid with hybridization probe.

Also can by with the dna immobilization of examine at solid support for example on the nylon membrane, and make the probe of mark and this sample hybridize to detect target nucleic acid.Similarly, also can be by with mark probe-immobilized on solid support, and the sample that hybridization comprises the target nucleic acid of mark detects target nucleic acid.Washing with after removing nonspecific hybridization detectable label.

Also can contact to allow by the probe of the cell or tissue sample that will thoroughly change and mark to hybridize with target nucleic acid and detect in position target nucleic acid.After washing is with the probe of removing non-specific binding, detectable label.

These assay methods can be that direct hybridization assays maybe can comprise sandwich assay, and it comprises and use a plurality of probes, as at U.S. Patent number 5,681, and 702,5,597,909,5,545,730,5,594,117,5,591,584,5,571,670,5,580,731,5,571,670,5,591,584,5,624,802,5,635,352,5,594,118,5,359,100,5, summarize in 124,246 and 5,681,697, each patent is incorporated herein by reference.

Various hybridization conditions be can use, above-mentioned high stringent condition, medium stringent condition and low stringency condition comprised.Can under the stringent condition that only allows the hybridization of probe and target, measure.Can control stringency by changing such step parameter, described step parameter is thermodynamic variable, includes, but are not limited to temperature, methane amide concentration, salt concn, chaotropic salt concentration pH or organic solvent concentration.

Can carry out hybridization by the whole bag of tricks.Can be simultaneously or add successively the component of reaction with different order.In addition, reaction can comprise other reagent of many kinds.These reagent comprise salt, damping fluid, neutral protein such as white protein, stain remover etc., and described reagent can be used for helping best hybridization and detects, and/or reduce non-specific interaction or background interaction.Depend on the preparation method of sample and the purity of target, in the time of suitably, also can use the reagent that improves in other respects the effect of measuring, for example the inhibitor of the inhibitor of proteolytic enzyme, nuclease and biocide.

A. diagnosis

The method that the present invention also relates to diagnose, it comprises the differential expression level of disease association miRNA in biological sample that detect.Sample can derive from the patient.The diagnosis of patient's morbid state makes it possible to prognosis and selects therapeutic strategy.In addition, can come the etap of cell is classified by determining the miRNA molecule that interim (temporarily) expresses.

Can carry out the in situ hybridization of probe and the tissue array of mark.When the finger printing between individuality and the standard relatively, those skilled in the art can be based on finding make diagnosis, prognosis or prediction.It is also understood that the gene of indication diagnosis can be different with the gene of indication prognosis, and the characterization of molecules analysis of cell situation (molecular profiling) can be distinguished situation or the measurable result of responsive situation or refractory.

B. drug screening

The present invention also relates to screen the method for therapeutical agent, it comprises that the pathological cells that can express disease association miRNA contacts and estimate drug candidates to the effect of the expression overview of disease association miRNA with candidate therapeutic agent.Behind the miRNA that identifies differential expression, can carry out various mensuration.Can just regulate the ability screening test-compound of the genetic expression of disease association miRNA.Adjusting comprises increase and the minimizing in the genetic expression.

Test-compound or drug candidates can be any molecules of being checked with regard to the ability that changes directly or indirectly the expression of disease phenotype or disease association miRNA, for example, and albumen, oligopeptides, little organic molecule, polysaccharide, polynucleotide etc.Drug candidates comprises many chemical species, for example has to surpass 100 and be lower than the little organic molecule of about 500,1,000,1,500,2,000 or 2,500 daltonian molecular weight.Candidate compound can comprise to interact with the albumen recurring structure and particularly forms the necessary functional group of hydrogen bond, and generally comprises at least amido, carbonyl, hydroxyl or carboxyl, preferably comprises at least two chemical functional groups.Candidate agent can comprise ring-type carbon (cyclical carbon) or heterocycle structure and/or aromatics or polycyclic aromatic (polyaromatic) structure that replaces with one or more above-mentioned functional groups.Also in biomolecules (comprising peptide, sugar, lipid acid, steroid, purine, pyrimidine, its derivative, analog or combination), found candidate agent.

Can be just in conjunction with the ability of disease association miRNA or regulate the combinatorial library of the potential conditioning agent of its active ability screening.Combinatorial library can be the set of multiple compounds, and described compound is that for example the chemosynthesis carried out of reagent or biosynthesizing produce by making up many chemical structures unit.The preparation in combinatorial chemistry library and screening are known for a person skilled in the art.The peptide library of the peptide that these combinatorial chemistry libraries include, but not limited to encode, benzene phenodiazine

Class, diversomers be hydantoins, benzene phenodiazine for example

Class and dipeptides, vinylogous polypeptide, the similar organic synthesis body of little library of compounds, few carbamate (oligocarbamates) and/or peptidyl phosphoric acid ester, nucleic acid library, peptide nucleic acid(PNA) library, antibody library, carbohydrate library and little organic molecule library.

11. gene silencing

The present invention also relates to reduce with nucleic acid of the present invention the method for the expression of target gene in cell, tissue or organ.Can reduce (it comprises basically the sequence with one or more binding site complementations of said target mrna) expression of target gene by expressing nucleic acid of the present invention.Described nucleic acid can be miRNA or its variant.Nucleic acid also can be can be through processing to produce pri-miRNA, pre-miRNA or its variant of miRNA.The miRNA that expresses can with said target mrna on basically complementary binding site hybridization, this can cause the activation of the gene silencing of RISC mediation.Utilizing the example of research of the overexpression of miRNA is the people 2004 such as Yekta, Science304-594, and it is incorporated herein by reference.Those skilled in the art recognize that by using antisense method well known in the art and U.S. Patent number 6,506,559 and 6,573, the RNAi method of describing in 099 (it is incorporated herein by reference) can be used for nucleic acid of the present invention to suppress the expression of target gene.

The target of gene silencing can be the albumen that causes the second albumen silence.By suppressing the expression of target gene, can increase by the second protein expression.The example of the establishment that miRNA expresses is the people 2004 JBC 275-52361 such as Esau; The research of carrying out with people 2005 Nucleic Acids Res.33-1290 (it is incorporated herein by reference) such as Cheng.

12. gene strengthens

The present invention also relates to use nucleic acid of the present invention to increase the method for the expression of the target gene in cell, tissue or the organ.Can increase (it comprises basically the sequence with pri-miRNA, pre-miRNA, miRNA or its variant complementation) expression of target gene by expressing nucleic acid of the present invention.Described nucleic acid can be anti--miRNA.Anti--miRNA can be hybridized with pri-miRNA, pre-miRNA or miRNA, thereby reduces its activity of gene expression.Also can increase by expressing nucleic acid of the present invention the expression of target gene, described nucleic acid of the present invention basically with target gene in the part of binding site complementary, the combination of described like this nucleic acid and binding site can prevent the miRNA combination.

13. treatment

The present invention also relates to such method, the method with nucleic acid of the present invention as with grow the dysfunction for example related disease of cancer or conditioning agent or the target of illness.Usually, the nucleic acid molecule of described request protection can be used as at least in part the Enhancer elements agent with described nucleic acid complementation.In addition, the miRNA molecule can be used as the target of method of screening therapeutical agent, for example the inhibition of miRNA molecule or activate adjustable ganglion cell's atomization, for example apoptosis.

In addition, can be with existing miRNA molecule as starting material, with the modified miRNA molecule of production sequence to modify it to for example target-specific of oncogene, wide spectrum drug resistant gene or another kind of therapeutic target gene.In addition, can modify the miRNA molecule, purpose is it is processed, and then it is produced as and double-stranded siRNA that can treatment-resistant cognation target.In addition, the miRNA molecule can be used for organizing reprogramming method (tissue reprogramming procedures), for example, can be by in different cell types or stem cell, expressing the clone that the miRNA molecule transforms differentiation.

14. composition

The present invention also relates to comprise nucleic acid of the present invention and the pharmaceutical composition of drug acceptable carrier randomly.Said composition can be used for diagnosis or therepic use.Can by known method drug administration composition, in described method, in external or body, nucleic acid be imported the target cell of wanting.Normally used gene transfer technique comprises calcium phosphate method, DEAE-dextran method, electroporation, microinjection, viral method and cationic-liposome.

15. test kit

The present invention also relates to such test kit, this test kit comprises nucleic acid of the present invention and any or all following materials: measure reagent, damping fluid, probe and/or primer and Sterile Saline or the acceptable emulsion of another kind of medicine and suspension substrate (suspension base).In addition, test kit can comprise the illustrative material that comprises be used to the guidance of implementing method of the present invention (for example, rules).

Embodiment 1

The prediction of miRNA

We use two kinds and Application No. 60/522,459,10/709, the similar computer approach of method that is used for prediction miRNA of describing in 577 and 10/709,572 (its content is incorporated herein by reference) is investigated with regard to the whole human genome of gene pairs of potential coding miRNA.In brief, scan the non-protein-coding region of whole human genome with regard to hairpin structure.Give the hairpin structure of prediction and potential miRNA scoring according to thermodynamic stability and structure and contextual feature.Use the miRNA calibration algorithm that has been verified in the Sanger database.

1. for the first time screening

Table 11A-11C shows the miRNA (" GAM title ") from the biology in sequence (" precursor sequence "), sequence flag symbol (" PRECUR SEQ-ID ") and the source of the hairpin structure of each prediction of the computer screening first time (" GAM is biological ") and prediction.Table 12 shows the sequence (" GAM RNA sequence ") of each miRNA (" GAM title ") and the biology in sequence flag symbol (" GAM SEQ-ID ") and source (" GAM is biological ") and Dicer cutting position (" GAM POS ").The hairpin structure of prediction and the sequence of miRNA are also listed in the table 10.

2. programmed screening

Table 1 has been listed the SEQ ID NO (" HID ") of each hairpin structure of predicting of for the second time computer screening.Table 1 has also been listed the genome position (" hairpin structure position ") of each hairpin structure.The form of genome position is series connection＜chr_id〉＜chain〉＜zero position 〉.For example, 19+135460000 refer to No. 19 karyomit(e) ,+chain, zero position 135460000.Karyomit(e) 23-25 refers to X chromosome, Y chromosome and Mitochondrial DNA.Chromosomal location based on by UCSC ( The hg17 of the human genome that http://genome.ucsc.edu) carries outAssembling, the hg17 assembling produces based on NCBI Build 35 the 1st edition and by international man's genoid order-checking alliance (International Human Genome Sequencing Consortium).

Table 1 has also been listed hairpin structure and whether guarded (" C ") in evolution.There is such option, namely has paper spare genome version.Identify that by using the phastCons data hairpin structure is (" Y ") or nonconservative (" N ") that guards.PhastCons data the measuring the evolution conservative of each Nucleotide in the genome of chimpanzee, mouse, rat, dog, chicken, the frog and zebra fish that be human genomes, it is based on phylo-HMM, phylo-HMM uses the in pairs comparison (pair wise alignment) based on (best-in-genome) best in the genome of each species of BlastZ, then carry out 8 genomic multiZ comparisons (people such as Siepel, J.Comput.Biol 11,413-428,2004 and the people such as Schwartz, Genome Res.13,103-107,2003).Scoring is at least 0.9 if the average phastCons that any 15 nucleotides sequences of 7 species in the hairpin structure stem list is conservative, hairpin structure is listed in conservative (Berezikov so, E. wait people Phylogenetic Shadowing and Computational Identification of Human microRNA Genes.Cell 120,21-24,2005).

Table 1 is also classified the genome type (" T ") of each hairpin structure as intergenic (" G "), intron (" I ") or exon (" E ").Table 1 has also been listed the miRNA of each prediction and the SEQ ID NO (" MID ") of miRNA*.Table 1 has also been listed the prediction scoring rank (" P ") of each hairpin structure in the numerical range of 0-1 (1 expression hairpin structure is the most reliable), such as people such as Hofacker, and Monatshefte f.Chemie 125:167-188, described in 1994.If rank is 0 or empty, then it is transformed into the PalGrade value of its less p-value＜0.05.Table 1 has also been listed the p-value (" Pval ") of calculating from the background hairpin structure of the value of each P scoring.As shown in Table, under a few cases Pval greater than 0.05.In each of these situations, hairpin structure high conservative or its are verified (F=Y).

As describing in detail in the table 2, table 1 has also listed whether by expression analysis (" E ") miRNA has been carried out verifying (Y=is that N=is no).Table 1 has also listed whether by order-checking (" S ") miRNA has been carried out verifying (Y=is that N=is no).If have sequence difference between the miRNA of prediction and the miRNA of order-checking, predict so the sequence of order-checking.Should be pointed out that the expression of failing to measure the sequence of miRNA or not detecting it represents that not necessarily miRNA does not exist.These undetected miRNA can express in the tissue except tested tissue.In addition, these undetected miRNA can express in tested tissue, but with experiment in stage of cell or different stage of situation or situation under express.

Whether table 1 has also been listed miRNA and has been shown such as table 2 at least a disease) in the differential expression (" D ") (Y=is that N=is no) that describes in detail.Table 1 also listed miRNA whether be present in 6.0 (in April, 2005) of (" F ") (Y=is that N=is no) Sanger DB Release ( Http:// nar.oupjournals.org/) in, as detected in people or mouse or in the people, predict.As mentioned above, listed miRNA is the component of prediction algorithm and the contrast of output in the Sanger database.

The gene location that table 1 has also been listed these hairpin structures clusters (genetic location cluster) (" LC "), and described hairpin structure is each other at a distance of within 5,000 Nucleotide.The total identical gene of each miRNA with identical LC cluster (genetic cluster).Table 1 has also been listed by mating fully by the seed of the 2-7 of miRNA the seed of its grouping (seed cluster) (" SC ") that cluster.Each miRNA with identical SC has identical seed.About the discussion of the seed length of 5 Nucleotide, referring to people such as Lewis, Cell, 120; 15-20 (2005).

Embodiment 2

The prediction of target gene

Then use and Application No. 60/522,459,10/709,577 and 10/709, describe the similar computer approach of method that is used for prediction miRNA in 572 (its content is incorporated herein by reference), the miRNA of the prediction that will screen from No. 2 computers of embodiment 1 is used for prediction target gene and its binding site.

1. the 1st screening

Table 13A-13C has listed from the target gene (" target ") of the prediction of No. the 1st computer screening and binding site sequence (" target binding site sequence ") and binding site sequence flag symbol (" target binding site SEQ-ID "), and the source of target biological (" target is biological ").Table 14 has been listed and the related disease (" disease name ") of target gene (" with the target gene of disease association ").Table 15 listed miRNA sequence flag symbol (" with the SEQ ID NOs of the GAMS of disease association ") and with based on the related disease of the miRNA of target gene (" disease name ").The sequence of binding site sequence is also listed in table 10.

2. the 2nd screening

Table 4 has been listed the target gene from the prediction of each miRNA (MID) of No. the 2nd computer screening and its hairpin structure (HID).The title of target gene is picked up from NCBI Reference Sequence release 9 (http://www.ncbi.nlm.nih.gov; The people such as Pruitt, Nucleic Acids Res, 33 (1): D501-D504,2005; The people such as Pruitt, Trends Genet., 16 (1): 44-47,2000; With the people such as Tatusova, Bioinformatics, 15 (7-8): 536-43,1999).The perfect complementary coupling of miRNA seed (site 2-8) by having 7 Nucleotide and the A (altogether=8 Nucleotide) on the UTR is identified target gene.About identifying the discussion of target gene, referring to people such as Lewis, Cell, 120:15-20, (2005).About being enough to miRNA is bonded to the discussion of the seed of UTR, referring to people such as Lim Lau, the people such as (Nature 2005) and Brenneck, (PLoS Biol 2005).

Then use target gene data set (producing by those target genes that only comprise the UTR that contains at least 30 Nucleotide) the prediction binding site that filters.Front 4000 Nucleotide of each UTR are only considered in the binding site screening, and when there is several transcript in each gene, consider the longest transcript.Filtration is reduced to 14239 with the sum of transcript from 23626.Table 4 has been listed the SEQ ID NO of binding site of the prediction of each target gene.When binding site was positioned at the mRNA of montage when upper, the sequence of binding site comprises 20 Nucleotide of 5 ' and 3 ' of binding site.Except those miRNA of the binding site that only has single prediction or those miRNA of having verified, the data in the table 4 are filtered, thereby only indication has those target genes of at least 2 binding sites.

Relation between the disease that table 5 demonstration miRNA (" MID ")/hairpin structure (" HID ") and its target gene cause.The title of disease is picked up from OMIM.About the discussion with the residing host gene of hairpin structure and disease-related principle even, referring to, Baskerville and Bartel, RNA, the people such as 11:241-247 (2005) and Rodriguez, Genome Res., 14:1902-1910 (2004).Table 5 has shown the number (" N ") with the miRNA target gene of disease association.Table 5 has shown also and the sum (" T ") of the gene of disease association that this number is available from the gene that has the binding site of miRNA through prediction.Table 5 has also shown the per-cent of N in T and the p-value (" Pval ") of hypergeometry analysis (hypergeometric analysis).Table 8 has shown the code of the disease in table 5 and 6.About the bibliography of hypergeometry analysis, referring to the Outline of Elements of Statistics II:Inferential Statistics of Schaum.

Relation between the disease that table 6 demonstration miRNA (" MID ")/hairpin structure (" HID ") and its host gene cause.We are defined as the hairpin structure gene on the complementary strand of host gene and are positioned on the gene: Intron_c represents that intron and Exon_c represent exon.We select complementary strand, because it can cause disease.For example, be arranged in the sudden change of the miRNA on the complementary strand.In the situation that two chains exist, when selecting intron and Exon_c intron, two kinds of situations are similar at miRNA.The logic of selecting is intron＞exon＞lntron_c＞Exon_c＞intergenic.Relation between table 9 display target sequence (" gene title ") and the disease (" disease code ").

Embodiment 3

The checking of miRNA

1. expression analysis-Di is 1 group

For determining hairpin structure and the miRNA in 0 prediction, we use and Application No. 60/522,459, the similar high-throughput microarray of method of describing in 10/709,577 and 10/709,572 (its content is incorporated herein by reference) detects expression in various tissues.For the precursor miRNA of each prediction, the ripe miRNA that check is derived from two stems of hairpin structure.

Table 2 shows the hairpin structure (" HID ") by the 2nd prediction group of the expression checking that detects relevant miRNA (" MID "), and detects therein the code (" tissue ") of the tissue of expressing.The tissue of table 2 and the code of disease are listed in the table 7.Some tissues in the tested tissue are clone.The lung cancer cell line (H1299) that has/do not have P53:H1299 has the P53 of sudden change.With the such construct transfectional cell series with P53, this construct is responsive to temperature type (having activity under 32 ℃).Under 32 ℃, test.

Table 2 also display chip is expressed scoring rank (500 to 65000 scopes).The threshold value of use 500 is eliminated non-significant signal, and by the scoring of standardizing from the MirChip probe signals of different experiments.Variation in the fluorescent material intensity between the experiment may be because the difference in RNA preparation or the mark effect causes.We standardize based on such hypothesis, and namely the total amount of the miRNA in each sample is relatively constant.At first our subtracting background signal from the original signal of each probe wherein is defined as 400 with background signal.Then, we multiply by 10000 with acquired results, again the background signal of add-back 400 with the average signal of each miRNA probe signals divided by all miRNA.Therefore, according to definition, the summation of the probe signals of all miRNA is 10400 in each experiment.

Table 2 has also shown the normalized Statistical Analysis of Signals (" Spval ") according to normalized score calculation.For each miRNA, we have used the relevant control group from the miRNA tabulation of completely prediction.Each miRNA has the internal reference of the probe that comprises mispairing.Relevant control group comprises such probe, and this probe has similar C and G per-cent (abs diff＜5%) to have similar T _mProbe signals P value is the ratio to the relevant control group probe with identical or higher signal.The result is p-value≤0.05, and scoring surpasses 500.Classify as at SPVal in 0.0 the situation, this is worth less than 0.0001.

2. expression analysis-Di is 2 groups

For determining that further we have detected expression in other tissue at hairpin structure and the miRNA of 0 prediction.Table 16 is by the following expression data of listing miRNA: HID: the hairpin structure sequence flag symbol of the sequence shown in the table 17; MID: the miRNA sequence marker character of the sequence shown in the table 17: tissue: tested tissue; S: chip expression scoring rank (scope=100-65000); Dis.Diff.Exp.: disease-related differential expression and therein to its tissue of testing; R: the ratio of disease-related expression (scope=0.01-99.99); And abbreviation: brain mixture A-suffers from the cerebral tissue mixture of Alzheimer; The mixture of all cerebral tissues of brain mixture B-; With brain SN-black substance.

3. order-checking

For further verifying for the second time hairpin structure (" HID ") of prediction, by using and Application No. 60/522,459, the similar sequence measurement of method of describing in 10/709,577 and 10/709,572 (its content is incorporated herein by reference) is verified many miRNA.Table 3 shows the hairpin structure (" HID ") by the sequence checking of the miRNA (MID) in the tissue (" tissue ") shown in the mensuration.

Embodiment 4

No. 19 chromosomal miRNA

MiRNA from the empirical tests in groups of embodiment 3 expresses at the placenta camber, and it has obvious sequence similarity, and is arranged in the homologous genes seat (Fig. 2) on the karyomit(e) No. 19.Scatter in the zone of about 100,000 Nucleotide of the miRNA of these predictions in the 19q13.42 locus.This genome area does not have the gene of proteins encoded and seemingly intergenic.The further analysis of genome sequence comprises the output of the prediction algorithm of overhauling us, has disclosed the dependency miRNA that more infers, and it is about 25 that mir-371, mir-372 and mir-373 are positioned at this downstream, zone, 000bp.54 miRNA precursors of inferring have altogether been identified in this zone.This miRNA precursor can be divided into 4 dissimilar relating sequences (Fig. 2).About 75% miRNA is highly related and is marked as the A type in clustering.Other three kinds of miRNA types, type B, C and D are comprised of 4,2 and 2 precursors respectively.Other 3 miRNA precursors (S1 to S3) of inferring have irrelevant sequence.Enjoyably, all mir-371, the mir-372 with adjacent is identical with the direction of mir-373 miRNA precursor for the direction of all miRNA precursors.

Further sequential analysis discloses in the zone of about 600bp that most of A-type miRNA are embedded in the middle repetition 35 times of clustering.This tumor-necrosis factor glycoproteins does not appear in genomic other zones, and only conservative in primate.This repeating unit is almost always surrounded by the upstream and downstream Alu repeated sequence.This clusters with the MC14-1 that extremely lacks Alu repeated sequence and has formed distinct contrast.

Fig. 3-A shows the comparison of the sequence of 35 repeating units that comprise A-type miRNA precursor among the people.This Identification the zones of 2 performance highest serial similaritys.A district inclusion A-type miRNA is positioned at 3 ' zone of tumor-necrosis factor glycoproteins.The 2nd zone is positioned at about 100 Nucleotide places, A-type miRNA precursor upstream.Yet the 2nd zone do not show high similarity in the tumor-necrosis factor glycoproteins unit of chimpanzee, and the zone that comprises A-type miRNA precursor shows high similarity (Fig. 3-B).

As if the zone that inspection comprises A-type tumor-necrosis factor glycoproteins shows, more variable than other zones of the miRNA of maturation by the 5 ' zone (5p miRNA) of the miRNA of 5 ' the stem coding of precursor.Mutability the 3 ' district (3p miRNAs) of the miRNA of this and the maturation of deriving from 3 ' stem is complementary.As expected, the ring region height is variable.Also can in the multiple sequence comparison of all 43 A-type miRNA, observe identical phenomenon (Fig. 4).

The comparison of the multiple sequence that represents among Fig. 4 has disclosed the discovery of following ripe miRNA about prediction.5p miRNA can be divided into 3 groups.Nucleotide 1 to 6 is for being rich in C/T, and it is relatively variable, and in most of miRNA by the CTC motif mark in the Nucleotide 3 to 5.Nucleotide 7 to 15 is rich in A/G, and except Nucleotide 7 and 8, it is that most miRNA are common.Nucleotide 16 to 23 is rich in C/T, and shows that again it is guarded in the member.Generally speaking, the 3p miRNA of prediction shows higher conservative property in the family member.Most of from the AAA motif, but minority has possibility vital 5 ' different sequence in its target identification.Nucleotide 8 to 15 is rich in C/T and shows high conservative.Last 7 Nucleotide conservative propertys are slightly low, but it comprises the GAG motif that most of members have in Nucleotide 17 to 19.

Repeating unit 5 ' zone Analysis and Identification potential hairpin structure.Yet in most of repeating units, these hairpin structures are preserved, and also come a howler from the effort of the hairpin structure clone miRNA of the highest scoring.There are 8 A-type precursors in long repeating unit, not finding.Do not demonstrate and A-type repeating unit or and the similarity of any other genome sequence around the sequence of these precursors.For 5 precursors in these A-type precursors, there is the Alu repeated sequence in the downstream that is positioned at significantly close A-type sequence.

Other miRNA types show following feature in clustering.Found 4 B group miRNA in the repeat region of about 500bp, one of them is positioned at the end that clusters.Two D-type miRNA, it is each other at a distance of about 2000 Nucleotide, is positioned at the section start that clusters and is included in the repeat region of 1220 Nucleotide.Enjoyably, two D-type precursor phases together.Among the miRNA of 3 dereferenced sequences 2, S1 and S2 just in time are positioned at after 2 D type miRNA, and the 3rd between A34 and A35.Usually, the zone that comprises these whole about 100,000 Nucleotide that cluster is repeated element and covers.This comprises for the repeating unit that comprises miRNA of this regiospecificity and is dispersed in a large number the repeat element of this genome range in clustering.

Embodiment 5

The clone of the miRNA of prediction

For further verifying the miRNA of prediction, the similar method of the method for describing in use and the Application No. 60/522,459,10/709,577 and 10/709,572 (its content is incorporated herein by reference) is cloned the many miRNA that describe among the embodiment 4.In brief, the miRNA for each prediction designs specific capture oligo.Use described oligonucleotide to catch, clone the specific miRNA in the library of deriving from the placenta that is rich in little RNA and it is checked order.

We have cloned 41 miRNA and D-type miRNA among 43 A-type miRNA, and 13 miRNA among the A-type miRNA do not present on initial microarray, but only carried out computer forecast.For 11 in the miRNA precursor of prediction, the ripe miRNA of 5p and 3p prediction is presented on microarray, and two kinds of miRNA have produced significant signal in all situations.Therefore, we attempt to clone 5 ' and 3 ' ripe miRNA in all clone attempts.For 27 among 43 clones' the miRNA, we can clone derived from 5 ' and the miRNA of 3 ' stem.Because it is not exhaustively that our clone makes great efforts, so may there be more miRNA precursor to encode simultaneously 5 ' and 3 ' ripe miRNA.

As viewed in many miRNA clone researchs (Lagos-Quintana 2001,2002,2003) (Poy 2004), permitted polyclonal miRNA in 3 ' the terminal unhomogeneity that shows.Enjoyably, we also observe unhomogeneity at the 5 ' end of a large amount of miRNA that clone.As if compare with the miRNA (6) that 3 '-stem is derived, this unhomogeneity is more general in the miRNA (9) that 5 '-stem is derived.In relatively, the unhomogeneity on 3 ' end and 3 ' with 5 '-miRNA (being respectively 19 and 13) that stem is derived similar.5 ' unhomogeneity mainly comprises the adding of a Nucleotide (mainly being C or A), but has added in one case 3 Nucleotide.MiRNA during this phenomenon clusters for No. 19 karyomit(e) is not specific.We comprise known miRNA and from observing this phenomenon (data do not show) among other chromosomal new miRNA in many other clones' miRNA.

Embodiment 6

The analysis that miRNA expresses

For further checking the expression of the miRNA of embodiment 4, we analyze the feature that miRNA expresses in several tissues with rna blot analysis.Total RNA and the use of using 40 μ g to separate at 13% denaturing polyacrylamide gel ³²The end-labelled oligonucleotide probe of P carries out rna blot analysis.Sequence oligonucleotide probe is 5 '-ACTCTAAAGAGAAGCGCTTTGT-3 ' (A19-3p, NCBI:HSA-MIR-RG-21) and 5 '-ACCCACCAAAGAGAAGCACTTT-3 ' (A24-3p, NCBI:HSA-MIR-RG-27).MiRNA is expressed as the long RNA molecule of about 22 Nucleotide, its have with in microarray analysis (the identical tissue specificity overview of tissue specificity overview of observing among Fig. 5-A).

How to transcribe in order to determine that MC19-1 clusters.A position that comprises the ESTs with polyadenylation signal and poly A tail has only been identified in the investigation of ESTs in this zone.This zone just in time is positioned at the downstream of A43 precursor.Only other zones with the ESTs that comprises polyadenylation signal just in time are positioned at after the mir-373, thereby show that mir-371,2,3 is positioned at independently on the transcript.We have carried out preliminary study, and described research concentrates on the zone of mir-A43, really are transcribed into the mRNA of poly-adenylylation to guarantee this zone.Use to cover the fragment that RT-PCR experiment that the primer in 3.5kb zone carries out causes obtaining to expect (Fig. 5-B).Use the total RNA of 5 μ g placentas and use few deoxythymidine to carry out the RT-PCR analysis as primer.With the following primer transcript that increases:

f1：5′-GTCCCTGTACTGGAACTTGAG-3′；

f2：5′-GTGTCCCTGTACTGGAACGCA-3′；

r1：5′-GCCTGGCCATGTCAGCTACG-3′；

r2：5′-TTGATGGGAGGCTAGTGTTTC-3′；

R3:5 '-GACGTGGAGGCGTTCTTAGTC-3 '; With

r4：5′-TGACAACCGTTGGGGATTAC-3′。True by the sequence verification fragment

The property.This zone comprises mir-A42 and mir-A43, and this shows that two miRNA are present on the identical primary transcript.

About the analysis of this further information of transcribing that clusters from 77 EST that are positioned at its inside.We find that 42 EST are derived from placenta.Because these ESTs are along the whole distribution that clusters, so it shows that whole clustering all express in placenta.This observation is consistent with the expression overview of observing in microarray analysis.Therefore, but these all miRNA in clustering coexpression all only has D-type miRNA exception, and it is unique miRNA at the HeLa cells.Enjoyably, in poly-family, 77 the EST neither ones and the miRNA precursor that are arranged in this zone are overlapping.This is with remove EST expressivity (EST representation) from transcript by Drosha processing consistent.

The inspection that microarray is expressed overview discloses miRNAs D1/2, A12, A21, A22 to have in several its hetero-organizations of being checked with A34 and is reflected as the different slightly expression overview that is low to moderate medium expression level.This can be by coding miRNA transcript alternative splicing or have the specific promotor of different tissues and explain along the existence that clusters by other.

The relatively announcement of the expression of the ripe miRNA of 3p and 5p for many miRNA precursors, is both expressed, but in most of the cases with different horizontal expressions.For most of pre-miRNA, 3p miRNA is to be higher than the horizontal expression of 5p miRNA.Yet, in 6 kinds of situations (mir-D1,2, mir-A1, mir-A8, mir-A12, mir-A17 and mir-A33), 3p and 5p miRNA are with similar horizontal expression, and in a kind of situation (mir-A32), 5p miRNA is to be higher than the horizontal expression of 3p miRNA.

Embodiment 7

Conservative property

Relatively announcement from the sequence of the sequence of the miRNA of all predictions of 4 types of embodiment 4 and other species (chimpanzee, macaque, dog, chicken, mouse, rat, fruit bat, zebra fish, fungi, Caenorhabditis elegans), in clustering, and all miRNA in the whole zone in fact, except in primate, do not guard.Enjoyably, this regional homologue is not present in any other genome of being checked, and comprises the genome of Mouse and rat.Therefore, this be the 1st for primate the specific and general miRNA that does not have for Mammals does not cluster.Homology analysis between chimpanzee and the people shows that all 35 tumor-necrosis factor glycoproteinss that carry A-type miRNA are (contiguous) of adjacency between two species.In addition, whole poly-family is seemingly identical between the human and chimpanzee.Therefore, a plurality of repetitions (multiple duplication) that cause MC19-1 to cluster occurring must chimpanzee and human separates (split) front occured and evolutionary process at each species in maintenance stablize.Should be pointed out that No. 19 karyomit(e) of known person comprises gene family and the large fragment that many series connection cluster and repeats people such as (, 2004) Grimwood.Therefore, clustering at MC19-1 aspect this is No. 19 chromosomal natural part.

In relatively, MC14-1 clusters and generally guards in mouse, and only comprises A7 and A8 miRNA in clustering, and except in primate, described miRNA is not (Seitz 2004) of guarding.On the contrary, all miRNA in the poly-family of MC19-1 are unique to primate.Investigation to all miRNA of finding in Sanger discloses, only have 3 kinds of miRNA, mir-198, mir-373 and mir-422a do not guard in mouse or rat gene group, yet, it is guarded in the dog genome, thereby and is not specific for primate.Enjoyably, and mir-373 clusters, and the mir-371 and the mir-372 that are positioned at the poly-family of MC19-1 downstream 25kb place be homology with A-type miRNA (Fig. 4) to a certain extent, but guards in rodent.

The relatively announcement of miRNA in A-type miRNA sequence and the Sanger database, and the homology of people mir-302 family existence maximum (Fig. 4-C).This homology is higher than the homology of observing with mir-371,2,3.Found mir-302 family (mir-302a, b, c and d) in closelypacked the clustering of 5 miRNA (comprising mir-367) that cover 690 Nucleotide, described family is arranged in the 1st intron of exon of the proteins encoded of HDCMA18p gene (searching number NM_016648) with the direction of antisense.Except the miRNA homology, there is not other homology to be present between mir-320 clusters and MC19-1 clusters.Mir-371,2,3 and mir-302a, b, c, d all be that the specific fact is noticeable for embryonic stem cell.

Embodiment 8

The differential expression of miRNA

The similar chip expression method of method of describing in the use and 0, use characteristic is extracted software (Feature Extraction Software) (version 7.1.1, Agilent) and is analyzed microarray images.Table 2 shows the ratio of the disease association expression (" R ") of comparing with healthy tissues.Table 2 also shows normalized Statistical Analysis of Signals (" RPval ").Be its medium tenacity with the signal setting of each probe.The variation range of strength of signal is the saturated level from 400 background level to 66000.Carry out the hybridization of 2 passages, and Cy3 signal and Cy5 signal are compared, wherein carry out (normally to disease) fluorescence and reverse chip (fluor reversed chip), probe signals is set to its average signal.By the described signal of standardizing with known miRNA average signal removal signal, thereby in each experiment or passage, the summation of known miRNA signal is identical.Calculate the signal ratio between disease and the healthy tissues.Signal ratio shows remarkable rise greater than 1.5, and the p value is 0.007, and signal ratio then has 0.003 P value greater than 2.In repeated experiments, estimating the P value based on these or larger signal ratio.

Differential expression analysis in the table 2 shows that significant change has occured in the expression of many miRNA in diseased tissue.Particularly, the MC19-1 miRNA of embodiment 4 differential expression in prostate cancer and lung cancer.In deriving from the cell of prostate cancer, the cognation of MC19-1 miRNA and cancer people 2003 such as () Dumur is supported in the evaluation of heterozygosity in MC19-1 zone forfeiture.

Claims (16)

1. the nucleic acid that separates, it is 12 to 250 Nucleotide in length, and comprise sequence with SEQ ID NO:3360, SEQ ID NO:13805, SEQ ID NO:13806, SEQ ID NO:13807, SEQ ID NO:13808 or SEQ ID NO:13809 have at least 60% identity at least 12 adjacency Nucleotide or formed by its complement.

2. the nucleic acid of the separation of claim 1, it comprises nucleotide sequence or its complement that has at least 70% identity with SEQ ID NO:3360, SEQ ID NO:13805, SEQ ID NO:13806, SEQ ID NO:13807, SEQ ID NO:13808 or SEQ ID NO:13809.

3. the nucleic acid of the separation of claim 2, it comprises nucleotide sequence or its complement that has at least 80% identity with SEQ ID NO:3360, SEQ ID NO:13805, SEQ ID NO:13806, SEQ ID NO:13807, SEQ ID NO:13808 or SEQ ID NO:13809.

4. the nucleic acid of the separation of claim 3, it comprises nucleotide sequence or its complement that has at least 90% identity with SEQ ID NO:3360, SEQ ID NO:13805, SEQ ID NO:13806, SEQ ID NO:13807, SEQ ID NO:13808 or SEQ ID NO:13809.

5. the nucleic acid of each separation among the claim 1-4, it comprises nucleotide sequence or its complement of SEQ ID NO:3360, SEQ ID NO:13805, SEQ ID NO:13806, SEQ ID NO:13807, SEQ ID NO:13808 or SEQ ID NO:13809.

6. the nucleic acid of each separation among the claim 1-5, its nucleotide sequence or its complement by SEQ ID NO:3360, SEQ ID NO:13805, SEQ ID NO:13806, SEQ ID NO:13807, SEQ ID NO:13808 or SEQ ID NO:13809 forms.

7. the probe that is formed by each nucleic acid among the claim 1-6.

8. the probe that is comprised of the Nucleotide of 8-22 adjacency at least, the Nucleotide of this adjacency and SEQ ID NO:13807, SEQ ID NO:13808 or SEQ ID NO:13809 are complementary.

9. the multiple probe of claim 8.

10. the multiple probe of claim 9, it is comprised of the probe that is called each miRNA complementation of differential expression in prostate cancer at least one and the table 2.

11. the multiple probe of claim 9, it is comprised of the probe that is called each miRNA complementation of differential expression in lung cancer at least one and the table 2.

12. comprise the composition of the multiple probe of claim 10 or claim 11.

13. comprise the biochip of solid substrate, described matrix comprises the multiple probe of claim 10 or claim 11, wherein on definite address, space each probe is attached to matrix.

14. the biochip of claim 13, the miRNA that is called differential expression in prostate cancer in wherein said probe and the table 2 is complementary.

15. the biochip of claim 13, the miRNA that is called differential expression in lung cancer in wherein said probe and the table 2 is complementary.

16. detect the method for the differential expression of disease association miRNA, its be included in the biological sample measure and SEQ ID NO:13806, SEQ ID NO:13807, SEQ ID NO:13808 or SEQ ID NO:13809 in any has the level of the nucleic acid of at least 70% identity

Its neutralization contrast is compared, the difference indication differential expression on the nucleic acid level.

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