CN102272330A - Methods and compositions diagnosing lung cancer, determining prognosis, and improving patient survival - Google Patents

Abstract

The methods and compositions diagnosing lung cancer, determining prognosis, and improving patient survival based on the levels or gene status of certain microRNAs (miRNA), are provided. The compositions comprise a reagent for reducing miRNA level, and a use method used for facilitating survival of a patient is also provided.

Description

The method and the composition that are used for pulmonary cancer diagnosis, prognosis and raising survival rate

Technical field

The present invention relates to diagnostic method (as lung cancer), determine disease prognosis and improve patient's existence based on the disease of microRNA (microRNA) expression.

Background technology

Lung cancer is the first cause that the world causes cancer mortality.Similar patient's lung cancer may have different clinical effectivenesses, is difficult to prediction patient's prognosis.

Though molecular biological understanding increases to some extent to lung cancer in recent years, but still can't at length understand the potential molecular mechanism.Need accurate prognostic indicator clinically to determine high-risk patient, also need the methods of treatment of designing optimal targetedly.

All publications that this paper discloses, patent, patent application and public announcement of a patent application have been listed this paper in also as a reference.

Summary of the invention

The present invention be part based on discovery, the level that micro-RNA expression increases or reduces in the special cancerous tissue sample.Therefore, herein according to miRNAs and the corresponding miRNA gene expression dose in status, the diagnosing cancer method and the composition that provide, and definite cancer patients's prognosis (as lung cancer).This paper also provides the cancer (as lung cancer) of coming gene diagnosis and prognosis to judge with detecting probe method detection miRNA or corresponding miRNA.

On the one hand, presents has been set forth a kind of method, to determine that a people has lung cancer, comprising: a) lung tissue sample miRNA expression level, wherein, individual tissue be under a cloud be cancer, the b) expression level of miRNA and reference material relatively, and c) if sample is showed the changing features that has that has a miRNA at least, the interpretation individual has maybe may suffer from lung cancer.Lung cancer can be small cell lung cancer, adenocarcinoma of lung, or squamous cell lung carcinoma.The miRNA of at least one can be hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p, perhaps corresponding homology.This method also can comprise at least has changing features at three miRNA, wherein, has at least three microRNAs all to be selected from the hsa-miR-210 of composition, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p, or its corresponding homology.If hsa-miR-210 in the sample, hsa-miR-30a and hsa-miR-182 express to be increased and hsa-miR-486-5p, and hsa-miR-140-3p expresses reduction, shows that the individual suffers from maybe may cause lung cancer.Can pass through microarray analysis (for example, according to intensity of hybridization signal, or based on the ratio of hybridization signal) to determining the expression level of miRNA.The expression level of miRNA also can be analyzed by Northern blot, in situ hybridization, or quantitatively RT-polymerase chain reaction is determined.Detect miRNA and derive from the lymphoglandula sample, blood, serum, or respiratory tract swab.

In yet another aspect, a kind of method that presents is set forth is to determine that a people has lung cancer.Have the level of the changing features of a miRNA at least comprising analysis, determine that successively the individual has or have lung cancer.By deletion or the amplification of miRNA, or determine the variation of gene according to the variation of the gene copy number of miRNA.

In yet another aspect, presents has adopted system's detection of lung cancer, comprising at least one pair of primer or a plurality of probe, wherein every pair of primer, or each probe can detect in the sample of different miRNA, wherein can detect hsa-miR-210 at least about 50 percent primer and probe sequence, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p and its corresponding same miRNAs.

In yet another aspect, presents is set forth the method for system's detection of lung cancer, wherein, system comprises at least one pair of primer or a plurality of probe, wherein every pair of primer, or each probe can detect different miRNA or expression of gene state, at sample, the hsa-miR-210 that wherein has at least about 50 percent primer or probe to detect, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p and its corresponding same miRNAs.The expression that meets one of them miRNA changes prompting lung cancer.

In yet another aspect, presents uses at least one pair of primer or a plurality of probe structure manufacturing system to come detection of lung cancer, every pair of primer wherein, or each probe can detect in the different miRNA samples and wherein have at least primer or probe about 50% can detect hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p and its corresponding same miRNAs.

This document also uses a kind of method diagnosing patient, and the expression level by patients with lung cancer miRNA comes the diagnosing patient.This method can comprise: according to the miRNA or the homologous gene of table 1 or table 2, determine lung cancer differentiation rank.

In yet another aspect, the method that presents is set forth is determined squamous cell lung carcinoma patient's prognosis existence, comprising: a) expression level of detection of lung cancer tissue samples at least one miRNA, b) expression level of miRNA and the individual prognosis existence expression level of miRNA in the comparative sample and reference material, and c).Referring to hsa-miR-31 or corresponding homologous gene to miRNA.

In yet another aspect, presents is determined the squamous cell lung carcinoma patient prognosis method of lifetime, comprising analyzing miRNA or corresponding expression of gene in the squamous cell lung carcinoma sample, to compare with the contrast lung cancer sample, miRNA genetic expression is high or low closely related with postoperative existence.This miRNA is hsa-miR-31 or corresponding homology.Determining of miRNA expression level can be by Northern blot analysis, in situ hybridization, or quantitative real-time polymerase chain reaction.Detect miRNA and derive from the lymphoglandula sample, blood, serum, or respiratory tract swab.

In yet another aspect, one or more primers that presents uses or probe are to determining squamous cell lung carcinoma patient prognosis lifetime, one or more primer or probe can be used to miRNA in the test sample, and miRNA can be hsa-miR-31 or corresponding homology hsa-miR-31.

In yet another aspect, one or more primers that presents uses or probe are used to make up a kind of reagent or system and determine squamous cell lung carcinoma patient prognosis lifetime, one or more primer or probe can be used to miRNA in the test sample, and miRNA can be hsa-miR-31 or corresponding homology hsa-miR-31.

In yet another aspect, the method for presents is improved a lung cancer people living, comprises that using a kind of effective drug dose is reduced by at least a kind of miRNA level.MiRNA can be hsa-miR-31 or corresponding homologous gene, and miRNA can be hsa-miR-210, hsa-miR-30a, hsa-miR-182, or corresponding homologous genes.Reagent can be sense-rna or small molecule disturbance ribonucleic acid.This method can comprise: use a kind of effective drug dose and be reduced to rare two kinds of miRNA levels, miRNA can be hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p and its corresponding homologous gene.Use a kind of effective drug dose and be reduced to rare three kinds of miRNA levels, miRNA can be hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p and its corresponding homologous gene.

In yet another aspect, the medicine that presents adopts improves a lung cancer people living, and medicine can be sense-rna or small molecule disturbance ribonucleic acid, and effectively dosage reduces the miRNA level.MiRNA can be hsa-miR-31 or corresponding homologous gene, hsa-miR-210, hsa-miR-30a, hsa-miR-182, or corresponding homologous genes.

In yet another aspect, presents comprises the composition that pharmaceutical carrier and pack are carried, and composition can be sense-rna or small molecule disturbance ribonucleic acid, has reduced the level of miRNA, and effectively dosage reduces the miRNA level.MiRNA can be hsa-miR-31 or corresponding homologous gene, hsa-miR-210, hsa-miR-30a, hsa-miR-182, or corresponding homologous genes.

Unless otherwise prescribed, the present invention relates to all technology and have identical connotation with vocabulary with Institute of Science.Though method and materials similar or be equal to the invention that can be used for putting into practice described herein, being described as follows of suitable method and material.All publications, patent application, the full content that patent and other bibliographys are mentioned herein to mention that mode is included in.When clashing, this standard comprises definition, will control.In addition, material, method and example are illustrative, rather than only plan to limit.

The invention details is embodied in following appended figure and the explanation.Other functions of the present invention, object and advantage are apparent in describing, scheme and stating.

Description of drawings

Figure 1A is a sorter to 1F, can be used for distinguishing healthy tissues and pernicious pulmonary lesion.Sorter makes up based on principle component analysis (PCA) and SVMs (SVM) analysis.Round dot is represented cancerous issue, the contiguous cancer beside organism of cross bar representative.The lung cancer of all pathology hypotypes, 98.2% (127/132) can correctly diagnose (Figure 1A) in the training set, 92% (92/100) can correctly diagnose (Figure 1B) in the test set, comprising 60 pairs of squamous cell carcinomas (squama cancer), and the 43 pairs of gland cancer and 13 paired small cell lung cancer (SCLC) patients' tissue.To the squamous cell carcinoma patient, 93.3% (56/60) can correctly diagnose (Fig. 1 C) in the training set, and 96.7% (58/60) can correctly diagnose (Fig. 1 C) in the test set, wherein comprises 60 pairs of squamous cell cancerous tissues.To the adenocarcinoma of lung patient, 97.8% (45/46) can correctly diagnose (Fig. 1 E) in the training set, and 90% (36/40) can correctly diagnose (seeing Fig. 1 F) in the test set, wherein comprises 43 pairs of adenocarcinoma tissue.

What Fig. 2 described is squamous cell carcinoma, the non-supervision cluster of the microRNA of gland cancer and small cell lung cancer differential expression, the miRNA of differential expression is selected by SAM, left figure based on miRNA in the ratio of cancerous tissue with the other healthy tissues of corresponding cancer, right figure based on miRNA at the cancerous tissue absolute signal.60 routine squamous cell carcinomas have been analyzed, 43 routine gland cancer and 13 routine small cell lung cancer samples.These three pathology hypotype lung cancer can not clearly be divided into 3 groups in this research, it should be noted that the small cell lung cancer sample focuses on a group separately.

Fig. 3 A and 3B are the blue mayer survival curves of squamous cell carcinoma patient's Kapp.Survival curve shows that the patient is in test set 60 routine squamous cell carcinoma cases; Fig. 3 A) and test set (20 routine squamous cell carcinoma cases; Fig. 3 B) in, patient's survival rate that high-caliber hsa-miR-31 expresses is lower, and patient's survival rate height that low hsa-miR-31 expresses.Training set P value is 0.007, and test set P value is 0.001.

Embodiment

Presents is a basis, and the miRNA express spectra of the genome range of part Study uses the normal or canceration lung tissue sample of the freezing preservation of paired.Specifically, utilize the DNA oligonucleotide chip, the expression of microRNA, the miRNA that compares at the cancer sample expresses corresponding non-cancer sample.Five microRNAs are confirmed as or the tumor sample of overexpression or expression is compared, corresponding non-cancer sample.Some microRNA also is confirmed as having change various disease states at different levels.In addition, the discovery of miRNA at different levels and relevant overall disease free survival rate patients with lung cancer.

Therefore, method that presents provides and composition are assessed on the basis of the diagnosis and the disease (as cancer, as lung cancer) of prognosis, microRNAs at different levels or the corresponding miRNA gene in status.On the one hand, provide method and composition, to determine that a people has on the basis of a kind of disease (as lung cancer) some microRNA at different levels.In yet another aspect, cancer patients's's (as patients with lung cancer) that method of providing and composition classify pathological forms is arranged, the differentiation of level, on the basis of neoplasm staging, some microRNA at different levels.In yet another aspect, particularly microRNAs at different levels are arranged on the basis of a definite prognosis existence human cancer (as lung cancer) of method of providing and composition.The patients with lung cancer and the drug ingredient of the improvement existence of method, being used for this method yet provides the same, and system and tool kit can be used for method described herein.

Because " a " that uses herein, " an " and " " may mean single or plural (promptly may mean one or more) unless otherwise indicated.

" individual " is meant vertebrates (for example, Mammals as the mankind) as what use herein.Mammals includes but not limited to the mankind, inhuman primate, ox, sheep, pig, horse, dog, cat, rabbit, guinea pig, hamster, gerbil jird, mouse, rat.In some embodiments, a people can be a people.Embody at some, a people can become a kind of disease of a Research of Animal Model for Study, as lung cancer.This is to be understandable that, when the individual is not the people, can determine herein in corresponding microRNA homology or the human microRNA of vertical family.

The individual can be a sex.A people may or may not can demonstrate any pathology phenotype lung cancer.Embody at some, a people can have the lung cancer of family's medical history.

So-called " lung tissue sample " is meant that tissue sample is from lung cancer.Tissue sample can, for example, a fresh sample, freezing sample, or retain sample, (preserving sample or paraffin embedding sample) as formalin.As described below, and according to concrete method, tissue can be used for all maybe can being subjected to one or more methods decomposition samples and becomes fritter, cell aggregate, or individual cells.

Lung cancer, comprising but be not limited to lung squamous cancer, small cell lung cancer (nonsmall-cell lung cancer) and adenocarcinoma of lung.

MiRNAs: microRNA

Length of nucleotides that microRNA (microRNA) is the noncoding RNA of strand, normally about 22 (for example 19,20,21,22,23,24 or 25).By part or all of sequence homology, can (Bartel, Cell 2004,116:281-297) with the said target mrna molecule of the 3-of microRNA end non-coding region (3-holds non-coding region).Interaction between the miRNAs and target gene can stop translates into proteinic gene.In some cases, when the coupling fully of target and miRNA, the degraded of gene also may take place.

Understanding microRNA can find at http://miRNA.sanger.ac.uk/ usually on the net.The problem of database is seen Griffths-Jones et al., Nucleic Acids Research, 2006, Vol.34.The microRNA implication is cell processes widely, as cytodifferentiation, and the growth of cell and necrocytosis (Cheng et al, Nucleic Acids Res 2005,33:1290-1297; John et al, PLoS Biol.2004,2:e363).Has nearly 1000 microRNAs (Zamore and Haley in human genome, Science 2005, and 309:1519-1524), nearly the prediction of 1/3rd Human genome is possible miRNA target (Lewis et al, Cell 2005,115:787-798).The discovery of rna editing explanation microRNA can regulate and control more gene (Blow et al, Genome Biology 2006,7:R27).

Lung cancer that this part file discloses the level of microRNA, relevant (for example, directly or be inversely proportional to).For example microRNA is listed in table 1, comprising title, and the microRNA of sequence and chromosomal localization, and whether explanation is to raise or downward modulation lung cancer.The disease of method diagnosis can basis as lung cancer, for example, lists in table 1 at any microRNA of gene level or status.System described herein can be used for determining that the level of one or more microRNAs lists in table 1, and/or the one or more microRNAs that are used on the level basis of diagnosing are listed in table 1.

Just can reach acceptable susceptibility and specific detection though only detect by single microRNA,, if detect plural at least microRNA mentioned herein, then susceptibility and specificity can increase.For example, in certain embodiments, at least two, three, four or five microRNAs are used for determining whether patient suffers from lung cancer, can obtain definite result in the table 1.

In certain embodiments, in numbering: have at least the expression level of the microRNA of two (for example, have two at least, three, four or five) to determine in the listed order of 1-5.For example, listed serial number: 1-3 could determine in the microRNA of gene level or status, or the status of level or gene, and have listed serial number in two (for example, two or three) microRNAs at least: 4 and 5 could determine.Embody the status at some, or gene level has a microRNA to be selected from the sequence numbering that the sequence numbering of microRNA: 1-3 and at least one microRNA are selected from microRNA at least: 4 and 5 can be undetermined.In some cases, the status of level or gene, have the sequence numbering of two (for example, two or three) microRNAs at least: the sequence numbering of 1-3 and level: the 4 and the 5th could determine.Listing in table 1 at all microRNAs of some embodiment gene levels or status determines.

Determine in some homology ability described herein that embody one or more corresponding miRNA of level.The miRNA that the explanation of the miRNA of " corresponding homology " refers to herein is from the corresponding people's of non-human vertebrates miRNA.Corresponding homology have at least usually sequence signature about 50% (for example, have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) corresponding miRNA is described for the sequence identity.For example, the ID of the miRNA of a corresponding homologous sequence number: 1, can there be the sequence signature about 50% (for example, to have about 50%, 55% at least at least, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology) serial ID number: 1.

When the sequence of a miRNA is approximately for example pointed out to have at least, 95% (for example may determine a reference sequences, serial ID number: 1), its purpose is, the sequence of this miRNA is an identical reference sequences but the sequence of miRNA may comprise nearly 5 changes of per 100 nucleotide sequences.These variations that reach 5 points may be deleted, and replace, and are additional, and may occur in order Anywhere, in one or more continuous sequences.

The specific identity nucleotide sequence between the sequence and the specific identity numerical order of reference sequences are determined as follows.At first, listed particular sequence identification number uses the BLASTZ of the standalone version of BLAST2 sequence (Bl2seq) program to contain homology version 2 .0.14 and BLASTP version 2 .0.14 in the sequence that nucleotide sequence compares.The BLASTZ of this standalone version on the World Wide Web in the NCBI website of United States Government (ncbi.nlm.nih.gov).Explain how to use the Bl2seq program can be at the appended BLASTZ of readme file.Bl2seq compares the homology of two sequences and uses or the BLASTP algorithm.Homology is to be used for the nucleotide sequence of comparison, and BLASTP is used for the aminoacid sequence of comparison.Compare two nucleotide sequences, set of options is as follows: I am the comparison (for example: C: seq1.txt) of first nucleotide sequence of comprising in a file; Being set to of-J comprises second nucleotide sequence in the file comparison (for example: C: seq2.txt);-p is set to homology; The neighbour is set to any filename of wanting (for example: C: output.txt);-Q is set to-1;-R is set at 2; All stay their default setting with every other option.For example, following order can be used for producing an output file, has wherein comprised relatively in two sequences: C: Bl2seq-i: seq1.txt--j: seq2.txt-p, blastn-o c: output.txt-q-1-r 2.Compare two aminoacid sequences, select Bl2seq to be set as follows: I am the comparison (for example: C: seq1.txt) of first aminoacid sequence of comprising in a file;-J is set to the comparison that a file is loaded with second aminoacid sequence (for example: C: seq2.txt);-p is set to blastp; The neighbour is set to any filename of wanting (for example: C: output.txt); Stay their default setting with every other selection.For example, following order can be used for generating an output file and comprises in the two seed amino acid sequences: C: Bl2seq-i: seq1.txt--j: seq2.txt-p, blastn-o c: output.txt-q-1-r2.The output text.If these two sequence homologies, designated document output will be introduced these homologous sequence couplings then.If these two comparative sequences are homology not, will there be concensus sequence in designated document output then.

In case coupling, Nucleotide that the position of coupling is identical or amino-acid residue are on these two sequences.The sequence identity is determined (for example to determine divided by the order of stipulating in the quantity of mating the also sequence by length, serial ID number: 1), or, multiply by the value 100 that is produced then by a length (for example, proposing a definite order) from continuous 20 nucleotide sequence collection.For example, nucleotide sequence has 20 couplings, listed sequential identification codes in the matching sequence: the 1st, and listed serial number: 1 (i.e. 20 ÷ 22*100=90.9) in identical 90.9 percent the sequence.It was noted that percent sequence signature value is rounded up to immediate 1/10th.For example, 75.11,75.12,75.13 and 75.14 is to be rounded to 75.1, and 75.15,75.16,75.17,75.18 and 75.19 is to be rounded to 75.2.People are also noted that this length value will be an integer forever.

Estimate the method for miRNA level

This method is on the basis of one or more microRNAs.Because in using, " level " refers to the number or the accumulation rate of molecule or its precursor of miRNA herein.This speech can be used to refer to the miRNA (intensity of hybridization signal of representative) in the sample of absolute quantity, or with the ratio of the miRNA of contrast (ratio of the hybridization signal of representative and miRNA contrast).Contrast can be the different miRNA from same sample, and its level does not change at the cancerous lung tissue sample, also can be from the same miRNA of different samples (as the cancerous tissue sample from same individual, or tissue samples another person does not suffer from lung cancer).

The molecule of the miRNA of one " precursor ", or " miRNA precursor " refer to crude miRNA genetic transcription, and about 70 Nucleotide that generally include a rna transcription thing are long.The precursor of miRNA is the activatory miRNA molecule handled of RNA enzyme (as Dicer, Argonaut, or RNAase III) normally, normally 19-25 (for example, 19,20,21,22,23,24, or 25) length of nucleotides.

The tissue sample of the miRNA level that " miRNA is at the lung tissue sample level " is meant.Though in most of the cases determine the miRNA level in the miRNA of lung tissue sample level according to directly measuring, the level of the miRNA that the level of imagination miRNA not only also can reflect at the lung tissue sample but also at other samples, for example at lymphoglandula sample (as lymphoglandula or lymph liquid), serum, blood or other biological fluid materials are as phlegm.The level of a miRNA can be at lymphoglandula sample (for example, section or lymphoglandula pin are inhaled) blood or serum, or determine on the basis of lung tissue swab.The level of a miRNA can be determined (for example analyzing with RT-PCR) from the sample of obtaining down through the endoscopic ultrasonography guiding.Endoscopic ultrasonography guiding fine needle aspiration (biopsy) down is a kind of non-operation sampling of minimal invasive techniques, thereby can carry out more detailed molecular marker analysis.MiRNA level in the working sample not only can be surveyed cancerous lung tissue, also can survey cancerous lung tissue other cancerous lung tissues in addition.For example, the serum sample miRNA level that can determine earlier, and follow-uply can carry out the analysis of regional lymph nodes miRNA.This multistep analysis may provide more information, increases the diagnosis confidence level.

The level of miRNA can be determined from each stage.For example, the level of a miRNA can be before operation, and in the operation, after the operation, before the oncotherapy, in oncotherapy, and/or oncotherapy was determined afterwards.The level of a miRNA can be determined on the basis of lung tissue swab.

The method of measuring the microRNA level comprises those known technology.For example, Northern hybridization, in situ hybridization, the RT-PCR technology, and/or microarray analysis is determined the level of miRNA.See Einat, Methods Mol.Biol.2006,342:139-157; And Thompson et al., Genes Dev.2006,20:2202-2207.

According to exemplary method, the purifying of cell total rna is at the cell centrifugation of nucleic acid extraction damping fluid.Precipitate nucleic acids, DNA enzyme are handled and are removed DNA and precipitation.Can separate at the gel electrophoresis sepharose according to standard technique RNA molecule, and be transferred to the nitrocellulose strainer, for example, the Northern hybridization technique.RNA can be fixed on the strainer heating then.Detect and quantize marker DNA or the rna probe that concrete RNA can use suitable complementary RNA.The autography detection probe can be with the hybridization exposure on film with microRNA.The densitometric scan film can provide a level of weighing the rna transcription thing accurately.In addition, rna transcription thing level can quantize at computerize imaging hybridization trace.

Except Northern and other nucleic acid hybridization techniques, the transcriptional level of RNA is weighed in situ hybridization.This technology relates in cell or tissue has radiolabeled nucleic acid probe (as cRNA probe) to the microcosmic cover glass with also formulating to exist with whole cell or tissue point.

The level of microRNA also can be determined in reverse transcription and amplified polymerase chain reaction (RT-PCR).The microRNA level can be with interior parametrization (for example, from being present in " look after the house " the mRNA expression level of gene of same sample).Suitable " house keeper " gene comprises myosin, glycerine triphosphoric acid desaturase (G3PDH) and people U6 as confidential reference items.Quantitative RT-PCR and variation are well-known common technology.The RT-PCR primer sees Table 1.In some cases, the microRNA of real-time quantitative PCR (qRT-PCR) technical Analysis may be than traditional tissue slice and more responsive at some early-stage cancer dyeing detection microRNA.The miRNA level that this qRT-PCR detects may provide responsive and special diagnosis, classification, and the instrument of prognosis lung cancer.On the one hand, presents provides miRNA the people's that the method for RT-PCR determines level (for example, a people has disease, as cancer).The miRNA level be to detect by qRT-PCR.In some cases, the horizontal used chip of microRNA detects.

Nucleic acid probe can also can use the method for chemosynthesis with method production described herein, is well-known technology.In addition, hybridization probe can the various detection labels of mark, for example, comprise radio isotope, fluorescent mark, reporter enzyme, vitamin H and other parts.This detectable label can amplify, and for example, available light-intensity method detects heat or light index substance.Marking method and this detector of detection are well-known technology.

Nucleic acid probe can be used for microRNA hybridization under the condition strict in the test sample.According to special detection, strict condition can be different, detect specific miRNA in a certain sample optimization.

Generally speaking, the stable dependence ionic concn and the temperature of hybridization.Generally, the condition severity of hybridization reduces, and secondly be to clean difference, but condition is higher, and is stricter.The hybridization conditions permission nucleic acid molecular probes of moderate strictness etc. are in conjunction with complementary nucleic acid molecule.Making nucleic acid molecular hybridization generally all has 60% identification (for example, have 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% at least, or 95% above identification) at least.

Low stringency condition hybridization refers to be meant uses 10% methane amide, 5xDenhart solution, and 6xSSPE, 0.2% SDS is hybridized in 22 ℃, and then at 1xSSPE, 0.2% SDS washs in 37 ℃.Gentle stringent condition is meant that using 50% methane amide, the solution of 5xDenhart, 5xSSPE, 0.2% SDS to hybridize at 42 ℃ uses 0.2xSSPE then, and 0.2% SDS is after 42 ℃ of washings.High severe condition are meant uses 50% methane amide, the solution of 5xDenhart, and 5xSSPE, 0.2% SDS is hybridized at 42 ℃, and the SDS at 0.1xSSPE and 0.1% washs in 65 ℃ then.Other gentleness and high severe condition that are fit to are well-known.For example, at Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989); And Ausubel et al., supra, 1999.Denhardt solution comprises 1%Ficoll, 1%polyvinylpyrolidone and 1% bovine serum albumin (BSA).20xSSPE (sodium-chlor, sodium phosphate, vinyl-amine tetraacethyl (ethylenediamine tetraacetic acid (EDTA))) comprises 3M sodium-chlor, 0.2M sodium phosphate and 0.025M (ethylenediamine tetraacetic acid (EDTA)).

In certain embodiments, one or more microRNA levels can be from detecting at more than one time point.This " serial " sampling can be fit to the monitoring of individual lung cancer progress.Serial samples can be carried out any time of wanting, as every half a year, and every year, every two years or more or less.The miRNA of measurement level and reference level can compare, every next new test sample, or relevant data level may be held less analysis.

To a certain specific miRNA, reference level generally are regarded as " normally ".The reference level basis is from the non-cancer lung tissue of same individuality miRNA level.Reference level do not have lung cancer miRNA level according to the individual.Reference level can be according to the population miRNA level of non-lung cancer.In some cases, reference level can be from one group of sample, just comprises in samples tested, can be in advance or definite sample test.

Being worth as " reference " used herein can be absolute value, relative value, and a value has a upper limit or lower limit, a series of value, mean value, an intermediate value, mean value, or ratio, especially control or benchmark value.

Reference value relatively may be listed in table 1 microRNA or its corresponding homology is carried out any (for example, one, two, three, four, or five).The comparison procedure of reference level and miRNA level can be suitable the categorical measures of any way for example carry out, when hybridization signal is used as the level of weighing miRNA, level can the qualitative relatively hybridization signal of intensity directly perceived.For quantitative measure, can compare by the numeric data of inspection and the data of statement (for example, checking figure) as histogram or line chart.In the process relatively manually (as visual), perhaps can be automatic.

In certain embodiments, relatively be the scale of determining between observed value and the reference level difference (for example, difference between observed value between comparison " doubly " or the per-cent and the reference level).Owing to use herein, " difference doubly " is meant the scale between miRNA observed value and the reference level difference.

MiRNA level in one " variation of characteristic " compares with reference level, can reduce significantly or roll up.Owing to use herein, the miRNA level that " increasing considerably " is meant increase is at least about 5% (for example, have 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% at least, or more than 50%).Equally, " significantly reduce " refer to the level that reduced miRNA at least about 5% (for example, have 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or 50% or more) as using herein.

Table 1 has been listed the variation of microRNA feature.The characteristics variation of one or more microRNA levels can be used as the diagnosing foundation.For example, have a kind of sequence numbering at least: the level of 1-3 microRNA can determine, has at least the number that can measure microRNA significantly to increase and can indicate lung cancer.Have a kind of sequence numbering at least: 4,5, have at least the number that can measure microRNA significantly to increase and can indicate lung cancer.When having a kind of sequence numbering: 1-3 at least, has a kind of sequence numbering at least: when the level of 4,5 microRNAs can determine, have at least the number that can measure microRNA significantly to increase significantly to reduce and to indicate lung cancer with rare one number that can measure microRNA.

In certain embodiments, all microRNA levels at different levels are listed in table 1, have at least one can measure sequence numbering: the number of 1-3 microRNA significantly increase with rare one can measure sequence numbering: 4, the number of 5 microRNAs significantly reduces can indicate lung cancer, have at least two can measure sequence numbering: the number of 1-3 microRNA significantly increase with rare two can measure sequence numbering: the number of 4,5 microRNAs significantly reduces can indicate lung cancer.

In certain embodiments, use more than one microRNA, but microRNA level and inconsistent suggestion or expression having diagnosing, is the suggestion of " majority " or the interpretation of result that expression can be considered.For example, utilize five microRNAs when this method, wherein three show lung cancer, and its possibility of result is regarded as hint or shows individual diagnosable lung cancer.But at least one or an a plurality of particularly microRNA characteristic change diagnosable lung cancer.For example, when microRNA is hsa-miR-210, the hsa-miR-210 that increases considerably may be prerequisite of diagnosing.

Estimate the method for miRNA gene state

It is the method for the diagnosing on basis that this paper also provides the gene (or its corresponding homology) that has a microRNA at least, lists in table 1.Have deletion or amplification in one the miRNA gene samples by analysis at least, can assess the gene state.Minimizing with respect to the miRNA gene in control sample one disappearance or the amplification can show that there is lung cancer in individuality.

By determining the comparison of lung tissue sample structure or sequence and control sample structure or sequence from doubtful individual cancerous lung tissue, the miRNA gene that can detect deletion or amplify.Be applicable to that any detection changes gene structure or the sequence technology can be used for present method.For example, the miRNA genetically deficient of existence and amplification can utilize the specific miRNA sequence of nucleic acid probe Southern hybridization to detect.Sequential analysis and single-strand conformation polymorphism analysis are also available.

The gene of the miRNA of disappearance or amplification also can use the polymerase chain reaction (PCR) of the gene of amplified fragments to detect, and can determine amplified fragments sequence or length by order-checking or electrophoresis if individual's dna sample is different from sample DNA.The gene elmination of miRNA can also be by closely-related miRNA the disappearance of gene chromosomal marker survey.

The gene copy number of individual miRNA gene in the cell at least one miRNA in also can be by the sample of measuring is assessed, beyond one of them gene copy number other two are the gene to any sex somatic chromosome or female sex chromosome miRNA, or the sex chromosome in more than one other miRNA genes is the male sex, can show that the individual has lung cancer.

Any technology that is suitable for detecting gene copy number is all available, comprises Southern hybridization and pcr amplification technology.The method of the another kind of lung tissue sample miRNA gene copy number of determining depends on a fact, and promptly many microRNAs or gene cluster and chromosomal marker or other genes are closely related.Losing of the chromosomal marker of heterozygosis or the closely-related gene of other genes illustrates that chromosomal marker or the closely-related gene of other genes have heterozygosity.The method skill this paper that measures the chromosomal marker of disappearance has also illustrated.

Can be used for determining miRNA gene other technologies, for example, the chip that allele-specific primers extends, the general array of polymerase chain reaction/low dose radiation, single base chain extension of microballoon, the inverting of sequence label molecular probe and composite sequence hybridization.

In certain embodiments, " control sample " can be that the people does not have the cancerous lung tissue sample one by one.In addition, control sample can be the set (for example, the known individual who does not have lung cancer) of a human organization sample.

Have at least the microRNA of (for example,, two, three, four, five) to list in table 1 or its corresponding homology could be determined the gene state.In the use of the more than one microRNA that is embodied in the gene state, but there be not consistent suggestion or the interpretation of result that can consider of expression of advising or representing to have " majority " of diagnosing.For example, utilize five microRNAs of gene when this method, wherein three show lung cancer, and its possibility of result is regarded as hint or shows diagnosing.Yet,, be diagnosed as the change that lung cancer requires one or more specific miRNA genes in some situation.

Method diagnoses the illness

The method that presents provides has a kind of disease (as cancer) with definite people, comprising: a) individual in the biological sample of definite at least one miRNA of level, and b) compare with reference level, the level that one of them characteristic changes, this shows disease.But medical diagnosis on disease include but not limited to, and cancer (as lung cancer, mammary cancer, esophagus cancer, cancer of the stomach, liver cancer, large bowel cancer, carcinoma of the pancreas, leukemia, lymphoma, kidney, bladder cancer, cervical cancer cancer, carcinoma of endometrium, ovarian cancer and carcinoma of testis), cardiovascular disorder (as coronary heart disease, hypertension and atherosclerosis), and with age diseases associated (as Parkinson's disease, Alzheimer's disease, diabetes).

For example, the individual of the method diagnosing that this document provides, comprising: a) definite at least one miRNA of level (for example, have at least in the table 1 of a miRNA listed, or corresponding homologous gene) the lung tissue sample from the individual, wherein, tissue be under a cloud be cancer, and b) compare with reference level, the level that one of them characteristic changes, what show miRNA is lung cancer.

In certain embodiments, a kind of individual of method diagnosing can comprise: 1) Bi Jiao reference level, have at least a miRNA (for example, have at least in the table 1 of a miRNA listed, or corresponding homologous gene) level, at least at the lung tissue sample of a miRNA from the individual, wherein, tissue be under a cloud be cancer, and b) determine whether the level of individual lung cancer based on changing features, have a miRNA's at least.This method can further provide and comprise the tissue sample of lung tissue sample from individual and/or isolated microRNA.

This document has also been imagined a kind of individual that the diagnosing of information is provided, comprising: a) listed in the table 1 of definite at least one miRNA of level, or corresponding homology, lung tissue sample individual, wherein this tissue canceration under a cloud, and b) diagnosing of miRNA of the level of information, wherein the conduct basis of the miRNA of one-level be provided, it is lung cancer that the level that diagnosing and one of them characteristic change has a tell-tale miRNA at least.

In certain embodiments, have at least the sequence numbering of the microRNA of (for example,, two or three): 1-3 to determine in level, and the number that increases considerably at least one measure microRNA and can indicate lung cancer.The sequence numbering that has the microRNA of (for example, one or two) in level at least: 4 and 5 determine, and the level that reduces significantly has a measurable indication microRNA lung cancer at least.(for example has one at least in level, one, two or three) the sequence numbering of microRNA: 1-3 and (for example have at least, one or two) the sequence numbering of microRNA: 4 and 5 could determine, and the number that increases considerably has a kind of sequence numbering of microRNA: 1-3 at least and the level that reduces significantly has a kind of sequence numbering of microRNA at least: 4 and 5 can show lung cancer.

In certain embodiments, list definitely in each microRNA table 1 at different levels, and the number that increases considerably has a kind of sequence numbering of microRNA: 1-3 at least, and in conjunction with the sequence numbering that has a microRNA on a large amount of levels that reduce at least: 4 and 5 can show lung cancer.In some cases, increase considerably the sequence numberings that have the microRNA of two (for example, two or three) at least at different levels: 1-3, in conjunction with the sequence numbering of a large amount of horizontal microRNA enzymes that reduce: 4 and 5 can show lung cancer.In some cases, increase considerably the sequence numbering of microRNAs at different levels: 1-3, in conjunction with the sequence numbering of the horizontal microRNA that reduces significantly: 4 and 5 can show lung cancer.

A tissue sample that changes miRNA at different levels also may reflect the miRNA gene in the variation.The gene state can reflect, for example, and the miRNA gene of deletion or amplification, or the gene of the miRNA by changing copy number.

Therefore, provide the individual of a method diagnosing at some, comprise that gene that the analyzing gene situation has a miRNA at least (for example, have at least the miRNA of a genes encoding to list in table 1) at the lung tissue sample under a cloud be canceration the individual, the corresponding miRNA genetic contrast sample that one of them characteristic changes the relative position of gene can show lung cancer.Deletion or the miRNA gene that amplified on the basis of deciding in the status that changes gene.In some cases, the variation of gene determines to have changed on the basis of status the miRNA gene of copy number.

This document also provides a method to diagnose individual lung cancer, comprises that the miRNA of the genes encoding of analyzing deletion or amplification miRNA lists in table 1, and miRNA genetic analysis wherein is at the lung tissue sample, canceration under a cloud.The miRNA gene of disappearance or amplification can show lung cancer with corresponding miRNA Gene Handling sample.For example, a kind of method can comprise analysis, has at least the miRNA of a corresponding miRNA gene to arrive listed sequential identification codes in the nucleotide sequence: 1,2,3 amplification, wherein Kuo Zeng miRNA gene can show lung cancer with corresponding miRNA Gene Handling sample.Can comprise analysis in a kind of method, have at least a corresponding miRNA gene to have listed sequential identification codes in the nucleotide sequence of a miRNA at least: 4,5 deletions have wherein been deleted the relative corresponding miRNA Gene Handling of miRNA gene sample and can have been shown lung cancer.In some cases, a kind of method can further provide and comprise the lung tissue sample of lung tissue sample cancer cells under a cloud from individual and/or isolated DNA.

This document also provides the individual of a method diagnosing, comprises that the miRNA that has a corresponding miRNA gene at least that determines gene copy number lists in the lung tissue sample of table 1 (or corresponding homologous gene), is from individual and canceration under a cloud.Other copy number surpasses that two miRNA gene is positioned at two kinds of sexes of karyomit(e) or sex chromosome is the women, and other miRNA genes that surpass are positioned at sex chromosome and are the male sex, can show lung cancer.For example, a method can comprise the listed serial number at least one microRNA in the corresponding miRNA gene that has at least of determining this gene copy number: the individual in the 1-3 sample, and copy number surpasses, and two genes to miRNA are positioned at two kinds of sexes of karyomit(e) or sex chromosome is the women, or the gene of more than one miRNA is positioned at sex chromosome for the male sex, can show lung cancer.In some embodiments, a kind of method can comprise the listed serial number at least one microRNA in the corresponding miRNA gene that has at least of determining gene copy number: the example in 4 and 5, and copy number is less than two and is the women for the gene of miRNA is positioned at two kinds of sexes of karyomit(e) or sex chromosome, or the gene that is less than 1 miRNA is positioned at sex chromosome for the male sex, can show lung cancer.Embody at some, a kind of method can further provide and comprise the lung tissue sample of lung tissue sample cancer cells under a cloud from individual and/or isolated DNA.

MicroRNA described herein also can be used for one or more as follows: the classification patients with lung cancer, forecasting risk, lung cancer, monitoring tumour progression patients with lung cancer, and on the basis of the patients with lung cancer of monitor therapy, at different levels or more microRNA is at the lung tissue sample, perhaps according to one or more microRNAs of gene status at the lung tissue sample.

Any method can further disclose the assessment that comprises recording or arrange the relevant individual situation of record (for example, by hand-written, computer, or audio means).For example, a method can comprise that whether the relevant information of record is the individual that the individual is confirmed as having lung cancer for prognosis existence, or the individual is categorized into specific lung cancer group.These steps will be discussed in more detail as follows.

Be used for determining the materials and methods of patients with lung cancer prognosis

The method that this part file provides is determined the patients with lung cancer of prognosis, comprising, for example, the individual of the existence prognosis of determining has the lung cancer method.The method of prognosis can be used for determining that a suitable course of treatment is for there being lung cancer individually.For example, the possibility of measuring survival can help to determine whether that more conservative or more radical method treats, and should consider, or therapeutic modality should merge.In addition, this prognosis can help to determine whether that for improving existence (as described) may be necessity and/or effective.

In certain embodiments, a kind of method, determine that prognosis existence has lung cancer to comprise individually: (a) number determine at least one miRNA cancerous lung tissue sample individual, (b) threshold level of the miRNA of the comparison of level in sample, level is wherein compared, the individuality of the dependency of the threshold value of miRNA or the relevant expection existence that is inversely proportional to.Because use " association " to be meant that the compare threshold of a low-level miRNA shows low chance for survival for lung cancer is arranged individually herein, vice versa.Because use " reverse correlation " to be meant that the compare threshold of high-caliber miRNA shows that surviving rate is low herein, vice versa.

Some method, and use this paper to describe relative threshold level on the basis that can comprise the miRNA level of determining that prognosis is survived.Starting point level can be by the method for any one diversification, but consequent threshold level provides first patient's the survival rate of the above-mentioned existence of miRNA to a certain degree to have the level of second group of patient miRNA of different survival rates to be lower than threshold level.

Threshold value can determine in order to following method, for example, and the one or more non-cancer cancerous lung tissue sample of the miRNA level of measurement.A starting point level can also have been determined miRNA at different levels individual lung cancer at a population by analysis.This can, for example, histogram analysis, the individual introduces in the test of whole formation, the miRNA of the level of a representative wherein, the survival rate individual of second representative.It is the specific miRNA that same or analogous level is arranged that two or more independently personal groups can estimate the formation of identifying the Ziren mouth.Determine that a threshold value then can be according to the level of miRNA, these different colonies of best differentiation, or the level of miRNA, the colony that best differentiation is different.For example, threshold value can be according to the mean level (ML) of the miRNA of mean value, and one group of survival rate of a collection of miRNA with high viability and mean level (ML) is low.A starting point level can also be represented two or more microRNAs of level.Two or more microRNAs can be represented, and for example, this ratio value is the level of each miRNA.

Threshold value can be a single numeral, the lung cancer that applied to everyone too, or according to the individual of specific subgroup.For example, old man may have different threshold values than young man, and the woman has and is higher than the different threshold level of man.In addition, threshold value can be a rank determine everyone.For example, threshold value can be the same individual in the non-cancer tissue of the level relatively of the miRNA in the cancerous lung tissue of a certain proportion of miRNA.

The possibility existence patients with lung cancer that the threshold level of verifying is distinguished is expressed and is lower than threshold level and expresses above-mentioned threshold value with the patient and can carry out single argument or use multivariate analysis.One or more variablees of the relation between the possibility that these methods can be used for determining and a specific result.In this case, the miRNA level of the dependency between the possibility that this method can be judged and the cancer patient of anosis or total survival rate.The method of any one diversification, well-known, those common skills are carried out these analyses and can be used.Example is the Kaplan-Meier method and the Cox regression model of univariate analysis.

Population be the mensuration threshold level (for example, passing through histogram analysis) on basis can to use the patient of formation be enough scales, with the patient who determines two or more independent groups different miRNA levels is arranged.Generally, such formation comprises having 25 examples (for example, have 25,30,40,50,60,75,100,125,150,200 at least, or surpass 200 examples) at least.Equally, a formation is verified and is determined that threshold level also has 25 examples (for example, have 25,30,40,50,60,75,100,125,150,200 at least, or surpass 200 examples) at least.

In addition,, have severally may have threshold value although single threshold value can independent two groups of patients, can independent most populations.For example, two different threshold values can the high-caliber miRNA of first patient from the miRNA of second group of patient's by-level, and from the miRNA's of the 3rd group of patient's low level.Just be enough to ban curve at many different threshold levels, as continuous lines, the possibility that it can be described, the patient of anosis or overall survival is as the miRNA level of a function.This curve can constitute the level of " a continuing " miRNA, the possibility there, and patient anosis or overall survival is the miRNA level that is directly proportional.Two or more miRNA levels can be represented such curve.

In certain embodiments, microRNA can be used in combination, to determine the cancer patient of existence prognosis provided herein.Two or more microRNAs that utilization combines can provide more prognosis meaning or prognosis.

The all right patients with lung cancer of miRNA level is anosis or a statistical index of overall survival, comprises pathological index (as the age, tumour size, tumor histology, clinical stages, family's medical history etc.).For example, the cancer at clinical stage may be that an anosis or total survival rate of statistics index can be according to different clinical stages with threshold value.Therefore, the different miRNA of threshold value can be used as the index of a function, adds up anosis or overall survival.

In some cases, the blue tracing analysis of Kapp can be used for determining the dependency between the level of survival rate and miRNA.

A kind of method can comprise: (a) definite level, have one the individual of miRNA in cancerous lung tissue at least, (b) the classification individual belongs to the individual's who is not first or second group lung cancer, wherein the miRNA of low at least one of first the individual level possibility that is listed in an existence increases, and second group individual high-caliber at least one miRNA.In some cases, having one miRNA at least can be hsa-miR-31.

Be determined and compared the microRNA level of threshold value according to one or more microRNAs at different levels in patient's sample, patient can be divided into one group of the possibility that certain anosis or overall survival are arranged.Patient is anosis or the possibility of overall survival can be assessed on this organizes the patient base of anosis or overall survival.

For example, a patient's sample can determine that a certain specific miRNA is low-level.Patient can be divided into one group of low-level miRNA then.If set up the possibility that the low miRNA of patient's expression level can increase anosis or overall survival, concrete cancer patients will be regarded as the possibility of anosis or overall survival.

Method described herein may further include the step of determining suitable therapeutic process for the individual.These technical ability may be different from the cancer patients who the estimates late stage prognostic indicator of surviving at the cancer patients who the estimates commitment prognostic indicator of surviving.For example, I phase prognosis may be the cancer patients towards the possibility of sustainable growth and/or the cancer of transfer, and IV phase prognosis patient can be towards virtuous methods of treatment treatment cancer.Therefore take definite suitable treatment to take into account these parameters.

In some cases, the measuring method of lung cancer for prognosis can comprise: (a) determine at least one miRNA in the level of cancerous lung tissue sample and (b) miRNA level and threshold level comparison, level is wherein compared, and the threshold value of miRNA is oppositely relevant with a people living.The miRNA that has one at least can be hsa-miR-31 or corresponding homology.

Described microRNA at different levels changes the microRNA gene state (as the microRNA of listing in table 1 and 2) that also may be reflected in.In certain embodiments, here provide one to determine to have individually lung cancer for prognosis existence method, the gene status (as the hsa-miR-31 or the corresponding homology of encoding gene) that has a miRNA gene comprising analysis at least, compare with check sample, show the chance for survival that the individual is higher or on the low side relatively.For example, provide a definite prognosis existence method, comprising the amplification of having analyzed corresponding miRNA gene hsa-miR-31, the miRNA gene of one of them amplification and the low survival rate of product associated cue in the same old way.In certain embodiments, provide a definite existence method of prognosis, comprised the copy number of determining the corresponding hsa-miR-31 of this miRNA gene, wherein the number of copies height is showing that surviving rate is low.

This paper also provides and has utilized probe, can survey microRNA level (or corresponding miRNA gene), and utilizes and comprise that one or more probes determine prognosis existence.For example, utilize one or more probes (or a system, comprise one or more probes), determining the prognosis of existence, the miRNA of its middle probe in can test sample, the existence of the dependency of the threshold level of miRNA or the measurable individuality of reverse correlation.One or more probes can be used for determining prognosis existence, the existence of the dependency of the threshold level of miRNA or the measurable individuality of reverse correlation, and wherein having a miRNA at least is hsa-miR-31 or corresponding homology.

In certain embodiments, provide one to utilize one or more probes to be used to make be defined as the surviving individual of prognosis of reagent or system lung cancer is arranged, the miRNA of its middle probe in can test sample, and level is therein compared the existence of the dependency of the threshold level of miRNA or the measurable individuality of reverse correlation.Provide one to utilize one or more probes to be used to make reagent or system, with the individual who determines the existence prognosis lung cancer is arranged, level is wherein compared, and the existence of the dependency of the threshold level of miRNA or the measurable individuality of reverse correlation wherein has a people hsa-miR-31 or corresponding homology at least.

Assist the materials and methods of medical treatment and professional person research

This document also provides method and material, assists medical treatment or researchist to suffer from lung cancer for determining whether a people.The medical professional can, for example, doctor, nurse, medical analyst and pharmacist.The researchist can, for example, investigator, investigative technique personnel, post-doctor researcher, undergraduate and graduate.Specialty can be assisted: the miRNA of the level that (1) is determined is in lung tissue sample individual and (2) communicate information, professional standards.

After miRNA level (or corresponding miRNA gene of status) report, medical professionalism can take to influence patient's the one or more action of nursing.For example, medical professionalism can write down the level of miRNA in patient's medical treatment record.In some cases, medical professionalism can record diagnosis lung cancer, or otherwise changed patient's medical treatment record, with reflection patient's medical condition.In some cases, whole medical recordss that professional medical can the review and appraisal patient, and assess multiple therapeutic strategy are the patient's of clinical intervention the state of an illness.

After obtaining patient's the level of miRNA, the healthcare givers can start or revise the data of treatment lung cancer.In some cases, the level of the miRNA of the level of miRNA and nearest circular in the report that medical professionalism can be former, and the suggestion change is treated.In some cases, the patient that can recruit medical speciality intervenes lung cancer in the new treatment of clinical trial.In some cases, medical professionalism can select to wait for, needs clinical intervention up to the patient symptom of begin treatment.

Medical personnel can be linked up with regard to the level (or corresponding miRNA gene) of miRNA with patient or families of patients.In some cases, the medical professional can provide a patient and/or the relevant information that gets with lung cancer of families of patients, comprising methods of treatment, and prognosis, and referral is to the expert, as the oncologist.In some cases, medical professionalism can provide a patient medical record for the expert, links up with regard to the level (or corresponding miRNA gene) of miRNA with patient or families of patients.

The personnel of research can use relevant miRNA level and/or the status information of miRNA gene, promote lung cancer research.For example, the researchist can compile data, the level of miRNA and/or the symptom of pharmacological agent lung cancer of the relevant curative effect in miRNA gene status, to determine a kind of effective treatment.In some cases, the level that the personnel of research can obtain a miRNA is estimated one and is gone into group, or continues to participate in research or clinical trial.In some cases, the seriousness that the personnel of research classify is based on one or more microRNA levels.In some cases, can study the level of miRNA of the relevant exercise question of specialty and/or the miRNA gene medical speciality of status.In some cases, can with the clinical evaluation lung cancer of medical professionalism, treat the symptom of lung cancer with reference to the theme of specialty research.

Any appropriate means can be used for information is conveyed to other people.For example, information can obtain directly or indirectly to the professional.For example, a Laboratory Technician's miRNA level can be input to a computer based record.In some cases, information conveyed is to make the medical treatment or the research record of actual change.For example, the healthcare givers can link up with regard to the medical treatment record with other medical professionals.In addition, the communication of any kind can be used for communicate information.For example, mail, e-mail, phone and aspectant interaction can be used.These information can also pass to the decision information of a specialty, the specialty that provides in the electronics mode.For example, information can pass on the information of a specialty to the database on the computer, and for example, this specialty can be obtained information.In addition, this information can convey to hospital, the clinic, or research institution is as procuratorial doctor.

For improving the material and the method for patients with lung cancer existence

This paper provides some microRNA level of using medicine to change (for example, increase or reduce) to improve the method for the existence of patients with lung cancer, comprises being listed in table 1 and table 2 microRNA.Any method that can increase or reduce the level of microRNA is provided herein.For example, can be used for the miRNA of inhibition of gene expression to include but not limited to, double-stranded RNA (for example, short chain or small molecules interference RNA or " RNA interference "), antisense nucleic acid, the RNA molecule of enzyme, as ribozyme, micromolecular compound and protein.These can be used for separately or with other combined utilization.Can reduce level (for example, by suppressing expression or the function of miRNA) or indirect (for example, the corresponding miRNA gene) of direct miRNA by influencing.

The method of improving individual lung cancer existence can comprise, for example, uses the level that effective dose has reduced miRNA, compares the individuality of the existence that is inversely proportional to the threshold level of miRNA.The manufacturing medicinal application effective dose of miRNA has reduced the level of miRNA, compares with the threshold level of miRNA, the individuality of the existence that is inversely proportional to.

In certain embodiments, the application effective dose has reduced the level of miRNA and has improved individual lung cancer existence, and the miRNA that has reduced level is selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, and corresponding homology.The manufacturing medicinal application effective dose of miRNA has reduced the level of miRNA, and wherein, miRNA is selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, and corresponding homology.Reduce hsa-miR-31, or reduce the one or more hsa-miR-210 of hsa-miR-31 bonded, hsa-miR-30a, hsa-miR-182 can be particularly useful.

Individual's (for example, described herein method) that method described herein may further include definite existence prognosis before.

In certain embodiments, the above level of microRNA can reduce, to improve lung cancer patient existence.Can accomplish this point, for example, a reagent has reduced two or more microRNA levels.In addition, two or more reagent can be used for reducing two or more microRNA levels.For example, provide a method, improved lung cancer existence, comprised that being reduced to rare two microRNAs is selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, its corresponding homology.Provide and utilize one or more manufacturing medicines to improve lung cancer existence, wherein be reduced to rare two microRNAs and be selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, its corresponding homology.In certain embodiments, provide the method that improves individual lung cancer existence, comprised that being reduced to rare three microRNAs is selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, its corresponding homology.Provide and utilize one or more manufacturing medicines to improve lung cancer existence, comprise that being reduced to rare three microRNAs is selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182, its corresponding homology.

In certain embodiments, in some cases, this provides a method for the survival that promotes patients with lung cancer, comprise that individuation uses the effective dose of one or more medicines, reduce hsa-miR-31, hsa-miR-210, the level of hsa-miR-30a and hsa-miR-182.In some cases, this is reduction hsa-miR-31, hsa-miR-210, and the level of hsa-miR-30a and hsa-miR-182 promotes the drug manufacture of the survival of patients with lung cancer should use a kind of or multiple composition that foundation is provided.

In certain embodiments, in some cases, here provide a medicine to form, comprise the medicament of the level that can reduce certain miRNA and a kind ofly controlling pharmaceutically acceptable carrier, wherein having a miRNA at least is hsa-miR-31, hsa-miR-210, hsa-miR-30a, or hsa-miR-182.In some cases, at least one miRNA is hsa-miR-31.In some cases, at least one miRNA is hsa-miR-210.In some cases, at least one miRNA is hsa-miR-30a.In some cases, at least one miRNA is hsa-miR-182.In some cases, this medicament can be double-stranded RNA (for example, short or siRNA, or " siRNA "), the antisense strand of nucleic acid, or RNA molecular ratio such as ribozyme with enzymic activity.Promote the method and the medicine of survival to describe in detail at this.

The situation of survival can be divided into two kinds: a kind of is the survival of no disease, and another kind is comprehensive survival.Do not have tumor recurrence and/or development after " no disease survival " refers to diagnosis of case, for example, a tumour does not have the patient of recurrence." survival " refers to the survival time behind the diagnosis of case, and no matter whether its tumor recurrence comprehensively.

In some cases, the expression of a specific miRNA can be disturbed inhibition by derivative RNA at this miRNA.It is isolatingly to have double-stranded RNA (dsRNA) product of at least 70% (for example at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) homology to realize with the part miRNA gene by adding that RNA disturbs.In some cases, this dsRNA molecule can be " short or siRNA " or " siRNA ".

The siRNA that can be effective to current these methods can be the short dsrna of 10-30 length of nucleotides (for example, about 12-28,14-26,16-24, or 18-22 Nucleotide).SiRNA can contain the sense-rna chain of a just RNA chain and a complementary pairing, and the two forms double-stranded according to Watson-Crick base complementrity pair principle by annealing.Positive-sense strand contains the essentially identical nucleotide sequence with target miRNA.The positive-sense strand of siRNA and antisense strand can be made up of two single stranded RNAs of complementary pairing, also can be connected together by two parts of a complementary element paired " hair fastener " structure by strand and form.

By insertion, disappearance, replacement and/or the conversion of one or more Nucleotide, siRNA may be different with the RNA that forms naturally.These changes comprise the adding (as being added in the terminal or inner of siRNA) of non-nucleotide material, cause siRNA can resist the modification of ribozyme digestion, perhaps the one or more Nucleotide among the siRNA are replaced with deoxynucleotide.In some cases, one of siRNA or two chains can comprise 3 ' protruding terminus.SiRNA can obtain by chemistry or biological method, and perhaps plasmid or the virus vector expression from reorganization obtains, and this will set forth hereinafter.

The expression of a specific miRNA also can be suppressed by antisense nucleic acid.At this, " antisense nucleotide " refers to and can pass through RNA-RAN or the interactional mode combining target of RNA-DNA RNA, thereby changes the active nucleic acid molecule of target RNA.The antisense nucleotide that is applicable to current method can be to comprise with miRNA to adjoin sequence complementary single-chain nucleic acid (as RNA, DNA, RNA-DNA mosaic, PNA and LNA).In some cases, antisense nucleic acid comprises with miRNA and adjoins the nucleotide sequence that sequence has 70% (for example at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) complementary pairing at least.In some cases, the length of antisense nucleic acid is approximately 10-30 Nucleotide (for example about 12-28,14-26,16-24, or 18-22 Nucleotide).

Antisense nucleic acid also can comprise the modification to nucleic acid backbone or sugar and base (or their Equivalent), thereby strengthens the relevant feature of target specificity, nuclease resistivity, transportation or other effects.These modifications comprise cholesterol half family, duplex inset such as acridine, perhaps one or more components with nuclease resistance.

Antisense nucleic acid can obtain by chemistry or biological method, and perhaps plasmid or the virus vector expression from reorganization obtains, and this will set forth hereinafter.

The nucleic acid that the expression of a specific miRNA also can be had enzymic activity suppresses.At this, " nucleic acid with enzymic activity " refers to the nucleic acid that contains the atural object calmodulin binding domain CaM, and this zone and miRNA adjoin the sequence complementary pairing, can shear miRNA specifically.In some cases, nucleic acid calmodulin binding domain CaM and the miRNA with enzymic activity adjoins the complementarity (for example 75-95% complementarity or 95-100% complementarity) that sequence has 50-100%.The nucleic acid of an enzymic activity also can have modification on base, sugar, phosphoric acid components.The nucleic acid with enzymic activity that typically can be used for current method is exactly ribozyme.

The nucleic acid of enzymic activity can obtain by chemistry or biological method, and perhaps plasmid or the virus vector expression from reorganization obtains, and this will set forth hereinafter.

Current have a lot of methods nucleic acid molecule can be imported in the cell, comprises cancer cells.These methods comprise microinjection, electroporation, liposome transfection, the transfection of calcium phosphate mediation, the transfection of deae dextran mediation, the micropartical blast technique is by colloidal dispersion transportation (macromolecular complex for example, gel particle, the water emulsifier, colloidal ion, mixed colloidal ion and liposome), and with antibody, linear gramicidins, artificial virus coat or other cell in carrier such as TAT coupling.

The nucleic acid medicament also can import in the external or intravital mammalian cell by known carrier in the document.Suitable carriers has virus vector and non-virus carrier, as plasmid vector.These carriers are helpful on the therapeutic dose that provides such as medicaments such as sense-rna or siRNA.

Advantage based on the system of virus is to import relatively efficiently allogenic nucleic acid in various cells.The suitable virus vector that imports nucleic acid has herpes simplex virus vector, vaccinia virus vector, cytomegalovirus carrier, mouse Moloney Leukemia virus carrier, adenovirus carrier, gland related virus vector, retroviral vector and slow virus.The taxis of virus vector can be controlled by using other viral envelope protein or surface antigen.For example, the related virus vector of gland can be made use of oral cavity vesicle virus, rabies virus, and Ebola virus, the surface protein of viruses such as Mokola is palmed off these virus.

Can contain any one inducible promoter or enhanser in nucleic acid or the carrier, thus can be by stimulating or add the expression that molecule is induced sense-rna or siRNA.These inducible systems comprise such as the tsiklomitsin inducible system, heavy metal inductive metallothionein promoter, the insect steroid hormone that moulting hormone or relevant steroid such as mouse sterone are replied, the mouse mammary tumor virus of steroid such as glucocorticosteroid and estrogen-induced, and temperature change inductive heat-inducible promoter.

If the dosage of a medicine can be enough to change the level (as reducing) of its target miRNA, this dosage can be described as the effective dose of this medicine so.In some cases, a kind of medicine can reduce the level of target miRNA 10% (for example, at least 10%, 20%, 30%, 40%, 50%, or greater than 50%) of miRNA level and Limiting Level differences at least.The typical doses of medicine provided herein (as nucleic acid drug) comprises the 0.1-3000mg/kg body weight, the 10-2000mg/kg body weight, and 50-1000mg/kg body weight, and 100-500mg/kg body weight, but be not limited to these scopes.In some cases, the dosage of medicine (as nucleic acid drug) is 100-500mg/g tumor weight (for example about 20-300mg/g tumor weight, 50-200mg/g tumor weight, or 100-150mg/g tumor weight).

Skill commonly used can be determined the suitable dose to individual one or more medicines in this field.Typical administration frequency comprises and only limits to that once per at least three weeks were once, and were whenever biweekly weekly in every month at least, weekly twice, on every Wendesdays time, on every Thursdays time, on every Fridays time, on every Saturdays time, or once a day, or more frequent.In some cases, the spacing of twice administration can be less than a week (for example being less than per six, five, four, three, two or one days).In some cases, the spacing of twice administration is a fixed.For example, can every day, per two days, per three days, per four days, per five days, per six days or be administered once weekly.In some cases, can be administered twice three times or more frequent every day.

A kind of administration of medicine can be secular, such as from about one month to by 3 years.For example drug delivery system can reach 2,3,4,5,6,7,8,9,10,11,12,18,24,30 or 36 months.In some cases, administration can not stop during administration.In some cases, the spacing of per twice administration can not be longer than a week.

Medicine described herein can be by any approach in this field to individual administration, comprises and only limits to intravenously, intraperitoneal, intraocular, intra-arterial, oral in the lung, in the alveolar, muscle, respiratory siphon, subcutaneous, in the meninges, stride skin, stride pleura, partial, suck (as spray), stride mucous membrane (as passing through nasal mucosa), pass through stomach, intraarticular, in the urethra, in the ventricle, internal rectum (as passing through suppository), intravaginal (as passing through vaginal suppository), in the skull, in the liver, and in the tumour.In some cases, administration that can general.In some cases, can topical.

Here also provide by controlling pharmacy acceptable carrier and can change the combination drug that the reagent of (as reduce) miRNA level is formed still.In some cases, medicinal composition comprises a kind of composition, and this composition can reduce the level of hsa-miR-210 or hsa-miR-30a or their homologue, and it can be siRNA, sense-rna or ribozyme.

In some cases, medicinal composition is aseptic.In some cases, medicinal composition is apyrogenic.The suitable pharmaceutically acceptable carrier of controlling has water, the aqueous solution, normal saline solution, 0.4% salts solution, 0.3% glycine and hyaluronic acid.Medicinal composition can also comprise traditional drug excipient and/or additive.Suitable drug excipient comprises stablizer, antioxidant, perviousness conditioning agent, damping fluid, pH regulator agent.Suitable additive comprises, and only limit to, the damping fluid that does not repel on the physiology, chelant (as DTPA and DTPA bisamide) and calcium chelated complexes (as calcium DTPA and CaNaDTPA bisamide), calcium salt or sodium salt (as calcium chloride, calcium ascorbate, calglucon and calcium lactate).Medicinal composition can be that liquid packaging also can be a dried frozen aquatic products.

For the solid pharmaceutical mixture, can use acceptable traditional non-toxic solid carrier on the pharmacopedics.Pharmaceutically acceptable solid carrier comprises other N.F,USP MANNITOL of pharmaceutical grade, lactose, starch, Magnesium Stearate, soluble saccharin, talcum, Mierocrystalline cellulose, glucose, sucrose, magnesiumcarbonate or the like.

MiRNA expression level and/or miRNA gene status detection system

The primer that comprises the miRNA that is used for detection table 1 and table 2 and system's (as real-time quantitative PCR (qPCR) primer, in situ hybridization, or microarray) of probe (as oligonucleotide) also are provided here.The system's (the qPCR primer was hybridization originally, or microarray) that comprises primer and/or probe can detect the situation of corresponding miRNA gene or their homologue.These systems in determining table 1 and table 2 miRNA or the expression level of their homologue and lung cancer evaluation in helpful.Some discussion here concentrate on the detection system of miRNA, but the people who is familiar with the skill commonly used in this field is readily appreciated that these descriptions are equally applicable to the gene status detection of miRNA.

Here the system that provides comprises primer and/or the probe that detects miRNA and/or detect its gene status.Some discussion here concentrate on the detection system of miRNA, but the people who is familiar with the skill commonly used in this field is readily appreciated that these describe major part and be equally applicable to comprise the primer of the variation (being referred to as the gene status of miRNA here) that detects genetically deficient, amplification and/or miRNA gene copy number and/or the system of probe.

At least system's (for example many primer mixtures) of the listed miRNA (or gene status of corresponding miRNA) of table 1 and table 2 can comprise one or more pairs of primers in the test sample, wherein every pair of primer specific miRNA in the sample (as biological sample) that can increase supposes the words that this miRNA exists in sample.

At least (the primer of quantitative PCR for example of the system of the listed miRNA (or gene status of corresponding miRNA) of table 1 and table 2 in the test sample, the probe of in situ hybridization and micro-array chip) can comprise many to primer and/or probe, wherein the every pair of primer and/or probe can detect the miRNA (or corresponding miRNA gene status) in the lung tissue sample, simultaneously, at least 15% (for example at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%) primer and/or probe can detect the listed miRNA of table 1 and table 2 or their homologue (or determine corresponding miRNA gene status).In some cases, a pair of primer and/or probe can detect miRNA different in the lung tissue sample (or gene status of corresponding miRNA).

In some cases, be used to diagnose system's (as microarray) of individual lung cancer can comprise many to primer and/or probe, wherein the every pair of primer and/or probe can detect the miRNA (or corresponding miRNA gene status) in the individual sample, simultaneously, at least 15% (for example at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%) primer and/or probe can detect table 1 and listed miRNA or their homologue (or gene status of corresponding miRNA) of table 2.In some cases, the pulmonary cancer diagnosis system can comprise at least one (as one, two, three, four or five) to primer, and wherein the every pair of primer can detect listed miRNA of table 1 and table 2 or their homologue, or the gene status of corresponding miRNA.In some cases, pulmonary cancer diagnosis system (as microarray) can comprise that at least one (as one, two, three, four or five) plant probe, and wherein every kind of probe can detect listed miRNA of table 1 and table 2 or their homologue, or the gene status of corresponding miRNA.In some cases, a pair of primer in the system or probe can detect the gene status (for example table 1 different miRNAs or the corresponding homologue listed with table 2, or corresponding gene) of different miRNA of lung group or different miRNA.Here the system that provides (as microarray) will be explained in detail hereinafter.

Here also provide multiple systems to be used for pulmonary cancer diagnosis.For example, use a system to carry out pulmonary cancer diagnosis, comprise in this system that at least one (as one, two, three, four or five) are to primer and/or probe (as oligonucleotide), wherein the every pair of primer or probe can detect listed miRNA of table 1 and table 2 or their homologue, or the gene status of corresponding miRNA.In some cases, a pair of primer in the system or probe can detect the listed different miRNA of table 1 and table 2 or the level of its corresponding homologue, or the gene level of different miRNA.Listed miRNA or its homologue at least one table 1 and the table 2, or the characteristic of corresponding miRNA expression of gene level changes and all may indicate lung cancer.

The method that system's (as be used for one or more pairs of primers of qPCR, be used for the probe mixture of in situ hybridization, or microarray) is used for pulmonary cancer diagnosis also is provided here.These systems are made up of primer and/or probe many, the every pair of primer and/or probe different miRNA (or corresponding miRNA gene status) in can test sample wherein, simultaneously, at least 15% (for example at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%) primer and/or probe can detect listed miRNA of table 1 and table 2 or their homologue, or the gene status of corresponding miRNA.At least one miRNA or its homologue, or the variation of the characteristic of corresponding miRNA expression of gene level all may indicate lung cancer.

Here also provide utilization to detect the primer of miRNA and the method for probe manufacturing system.In some cases, provide a method that one or more pairs of primers or probe (as oligonucleotide) is used to make the individual detection system of lung cancer here.Wherein the every pair of primer or probe can detect a listed miRNA of table 1 and table 2 or the level of its corresponding homologue, or the gene level of different miRNA.In some cases, provide a method that one or more pairs of primers or probe (as oligonucleotide) is used to make lung cancer detection system (as microarray) here.The wherein every pair of primer or probe different miRNA or the level of its corresponding homologue in can test sample, or the gene level of different miRNA, at least 15% (for example at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%) probe can detect listed miRNA of table 1 and table 2 or their homologue, or the gene status of corresponding miRNA.

Here the system that provides can comprise the primer of the identical miRNA of two or more detections.For example, (when system is microarray) in some cases, the probe in the microarray can be (can be two, three, four, five, six, seven or more multiple copied) of multiple copied.In some cases, a system can comprise the different probe that detects identical miRNA.For example, two or more primers may be incorporated into the zone of the difference (overlapping or not overlapping) of same miRNA.

The arbitrary primer or the probe that can detect the miRNA level can use.In some cases, primer or probe can be oligonucleotide.What wish to make sense be that some sequences of detecting miRNA change to some extent is acceptable.Therefore, oligonucleotide sequence (or their complementary sequence) may be different with miRNA sequence described herein.The people who is familiar with the technology in this field is readily appreciated that sequence variation can't the remarkably influenced oligonucleotide detects the level of miRNA.For example, when comparing with above-described method, the oligonucleotide molecules of homologous and change can be held the sequence homogeny of relative height.Here oligonucleotide sequence that comprises and miRNA described herein the have 40% at least sequence homology of (as at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or) greater than 95%.In some cases, oligonucleotide can comprise two portions, and first part is used for detecting miRNA, and second section is connected on the matrix.In some cases, second section may comprise non-specific sequence (as polyT) increases distance between complementary sequence and stromal surface.

Oligonucleotide in the system described herein can comprise DNA, RNA, PNA, LNA, associating wherein several and/or the associating after modified.Oligonucleotide can also comprise the skeleton after the modification.In some cases, oligonucleotide comprises at least 9,10,11,12,13,14,15,16,17,18,19,20, or more than 20 successive nucleotide fragments, these fragments are complementary or identical with miRNA sequence described herein.An oligonucleotide can comprise two or multiple this complementary sequence.In some cases, 5 ' or 3 ' end of oligonucleotide can have an active group (as amine), and oligonucleotide is connected on the matrix.

In some cases, system can comprise the primer of one or more pairs of qPCR (being also referred to as real time PCR (RT-PCR) or kinetics polymerase chain reaction).QPCR can be simultaneously detect and quantitatively dna sequence dna special in the sample, quantitatively be absolute copy number or proofread and correct with the DNA total amount that adds or other suppressor gene after relative amount.The key character of qPCR is exactly the DNA product that real-time quantitative increases along with circulation in amplification procedure.Quantitative methods comprises uses the fluorescence dye that inserts double-stranded DNA, and ought be the DNA oligonucleotide probe of fluorescigenic modification with complementary DNA hybridization.The method of qPCR is widely known by the people in this field.

In some cases, a system can comprise the one or more probes that are used for the one or more miRNA of in situ hybridization test set tissue samples.For in situ hybridization, the probe of a mark is used for special DNA of position tissue part or RNA sequence.Cell and tissue sample can pass through to handle the fixed target nucleotide sequence, thereby are convenient to and probe hybridization.Probe can be the DAN complementary strand or the complementary RNA (nucleic acid probe) of mark.Probe can be hybridized with target sequence under comparatively high temps, and the probe with surplus cleans up (will use the RNA enzymic hydrolysis earlier, in case the not superfluous rna probe of hybridization is arranged) then.By adjusting the parameter of solution,, remove the probe of any non-specific combination as temperature, salt and/or washing agent concentration.Probe can be used radioelement, fluorescence, antigenic mark, thereby can locate in tissue and quantitatively by radioautograph, fluorescent microscope or immunohistochemistry.In situ hybridization can also detect two or more sequences simultaneously by the probe of two or more marks.

In some cases, microarray that system can be a primer.Here " microarray " and " array " can be used alternatingly, and refer to the array (as orderly array) in combination (as the hybridization) site that comprises biological sample (target) supposition with unknown characteristics.In some cases, microarray refers to the set of specific oligonucleotide probe and is fixed on the position of determining on the matrix.

In some cases, a microarray can comprise multiple probe, wherein every kind of probe specific miRNA in can test sample, wherein at least 15% (for example at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%) probe can detect listed miRNA of table 1 or their homologue.

In some cases, provide the microarray that detects the corresponding miRNA gene status of miRNA here.The microarray that detects gene status has comprised known gene in this field.For example, a system can comprise the molecular inversion probes of sequence mark, is used to detect gene status.

Array can place on the matrix that paper, glass, plastics (as polypropylene, nylon, polystyrene), polyacrylamide, nitrocotton, silicon, optical fiber or other suitable solid or semi-solid material make, and forms the moulding of plane (as glass plate, silicon) or three-dimensional (as optical fiber, pearl, particle, micropore, kapillary).

In some cases, probe can be an oligonucleotide.The array that oligonucleotide forms by any several technical batterys on matrix.These technology comprise: (i) use photolithographic techniques original position synthetic (as high density oligonucleotide array); (ii) at medium mid point/seal low density probe on glass, nylon or nitrocotton; (iii) by shelter and (iv) the round dot trace on nylon or nitrocotton Hybond membrane.Oligonucleotide sequence also can be by magnetic bead or moving phase such as micropore or mode capillaceous and non-covalent being fixed on the matrix of anchor hybridization.

The famous technology that nucleic acid is connected on solid substrate such as the slide in several these fields is arranged.For example, one of them method be will amplification nucleic acid be blended into the matrix of modification or comprise can be in conjunction with the epi-position of solid substrate, have the group of positive charge as amino, aminoderivative or other, analogue in.Then amplified production can with solid-phase matrix (as slide) coupling.Solid-phase matrix is through acetaldehyde or other active group bag quilt, forms covalently boundly between they and amplified production, and amplified production has been connected on the slide with regard to covalency like this.The microarray that comprises amplified production can use such as Biodot (BioDot, Inc., Irvine, CA) (CEL Associates, Houston TX) makes the slide of point sample instrument and acetaldehyde bag quilt.Amplified production can be put on the slide of acetaldehyde bag quilt, also can process (Schena et al., Proc.Natl.Acad.Sci.U.S.A. (1995) 93:10614-10619) by reported method.Array also can be printed on glass, nylon (Ramsay by mechanical manipulator, Nature Biotechnol. (1998), 16:40-44), polypropylene (Matson et al., Anal.Biochem. (1995), 224 (1): 110-6) and on the silica gel sheet (Marshall and Hodgson, Nature Biotechnol. (1998), 16:27-31).Other the set array mode comprise electronic meticulous micro pipette (Marshall and Hodgson, supra) and the direct point sample of polynucleotide on the slice, thin piece of positive electricity bag quilt.Method such as using aminopropane base silicon face chemistry also is widely known by the people in this field, as describing to some extent on World Wide Web cmt.corning.com and cmgm.stanford.edu/pbrown/.

One of method of making microarray is by making highdensity nucleic acid array.Used technology is quick polynucleotide deposition (Blanchard et al., Biosenesors﹠amp; Bioelectronics, 11:687-690).Another method of making microarray be to use the method for sheltering (Maskos and Southern, Nucleic.Acids.Res. (1992), 20:1679-1684).In principle, above-mentioned any one array (as dot matrix on the nylon Hybond membrane) can use.Yet very little array has special advantages, because its hybridization system is very little, this will be approved by the professional in this field.

Test kit

This file also provides the test kit that is used for method described herein.In some cases, test kit comprises the system's (as one or more pairs of primers of qPCR, one or more probes of in situ hybridization, or microarray) that detects the miRNA level.In some cases, test kit can also additionally comprise the reagent that is used to detect.Test kit also comprises the specification sheets of the working method that detailed description is mentioned herein, and/or the network address with this class declaration is provided.

In some cases, test kit comprises the system's (as one or more pairs of primers of qPCR, one or more probes of in situ hybridization, or microarray) that is used for pulmonary cancer diagnosis described herein.Can comprise that in addition one or more control samples decide reference level, and/or how obtain the information of reference level.In some cases, test kit can also comprise that this test kit of use carries out the specification sheets of pulmonary cancer diagnosis.

In some cases, test kit can comprise the categorizing system (as one or more pairs of primers of qPCR, one or more probes of in situ hybridization, or microarray) of patients with lung cancer described herein.Can comprise that in addition one or more control samples decide individual segregation, and/or about the information of control sample, and in some cases, contain the test kit operation instruction of individual segregation.

In some cases, provide test kit here for patients with lung cancer survival prognosis.This test kit comprises primer and/or the probe that detects one or more miRNA (as table 1 and the listed one or more miRNA of table 2).In some cases, test kit can comprise the control sample that determines Limiting Level, and/or how to obtain the information of Limiting Level.In some cases, comprise that also with this test kit be the survive operation instruction of prognosis of patient.In some cases, test kit can comprise one or more reagent of change (as reducing) miRNA level, or comprises the medicinal composition of this reagent, thereby promotes survival.

The test kit of herein mentioning can also comprise some reagent, as substrate, marker, primer, the reagent of mark miRNA, the reagent that separates miRNA, hybridization and the feminine gender or the positive control that detect, pipe and/or other annex, the reagent of collection organization's sample, damping fluid, hybridizing box, cover plate or the like also might comprise software package (analyzing miRNA level and/or the variation of miRNA horizontal properties as using the statistical method mention herein) and be used to obtain any one password and/or the user name in aggregated data storehouse.

In some cases, test kit can comprise the medicinal composition of listed in change (as the reducing) table 1 a miRNA level, and promotes the operation instruction of patients with lung cancer survival with it.In some cases, test kit can also comprise one or more carriers or other reagent that is used for carrying medicinal composition.In some cases, test kit also comprises the operation instruction of medicinal composition.

This invention will be described in detail in following case, and this does not limit the application range of this invention.

Case 1---miRNA horizontal analysis sample is prepared

Patient and sample: the non-cancer lung tissue of 116 pairs of primary pulmonary cancerous tissues of picked at random and coupling.In this 116 routine sample, it is squamous cell carcinomas that 60 examples are arranged, and 43 examples are gland cancer, and 13 examples are small cell lung cancers.These samples all derive to treat as yet and just carry out operating patient.Sample is used liquid nitrogen flash freezer after cutting-out, and is stored in-80 ℃ (more than or equal to 5 years) always, up to extracting RNA.Have the former cancerous tissue of 20 routine lungs of trace information and the non-cancer lung tissue sample (storing at least 5 years) of coupling in addition and be used for confirming independently survival analysis.The lung sample peripheral portion that downcuts carries out paraffin embedding with conventional method, and section is then with phenodin and eosin dyeing.Two pathologists assess the concentration of tumour cell, and the histology of tumour has been carried out independently confirming.Trace information obtains from following the tracks of the register office.All samples all has clinical pathology information (smoking, age, sex, pathology hypotype, TNM classification, tumour stage, lymphoglandula stage, differentiation state and survivor's survival time after surgical operation).

The miRNA microarray is made: the design of miRNA microarray comprises 509 sophisticated miRNA sequences.Comprising the ripe miRNA of 435 people (122 prediction miRNA sequence (Xie et al. that comprise report from Britain Camb Wellcome Trust Sanger institute (network address http://microrna.sanger.ac.uk) registration, 2005)), article 196, the ripe miRNA of rat, 261 ripe miRNA of mouse.In addition, we have designed 8 does not have the oligonucleotide of homology with the RNA sequence, and makes up test kit (Cat.No.1550 with the miRNA primer of Ambion; Ambion, Inc., Austin TX) has in vitro synthesized their corresponding miRNA.These synthetic miRNA joins before analysis in the people miRNA sample with the difference amount as confidential reference items.

The design of all miRNA probe sequence all with the complete complementary pairing of total length of their corresponding sophisticated miRNA.For (China), we are connected to 40nt C6 5 ' the amido modified base of (3 ' terminal miRNA adds 5 ' terminal 19 poly-polyT) with probe sequence for CapitalBio Corp., Beijing on the slide that probe is easy to be fixed to the acetaldehyde finishing.(Ebersberg Germany) synthesizes oligonucleotide probe, uses EasyArray at MWG Biotech ^TMSampling liquid (CapitalBio Corp.) dissolving, concentration 40 μ M.Use SmartArray ^TMThree repetitions of each probe points of microarrayer (CapitalBio Corp.).

Target RNA mark: with Trizol reagent (Invitrogen; Carlsbad CA) extracts total RNA, and the miRNA separating kit with Ambion separates small molecular weight RNA then.According to the method (Thomson etal., 2004) of Thomson T4RNA ligase enzyme labelling method target-marking RNA.Briefly 2 T4RNA of unit ligase enzymes (New England Biolabs, Beijing, China) with 4 μ g small molecular weight RNA with 5 '-phosphoric acid-cytosine(Cyt)-uracil-cy3-3 ' (Dharmacon of 500ng; Lafayette, CO) mark.Labeled reactant carried out 2 hours at 4 ℃.The RNA of mark is with sodium-acetate and the 2.5 volume ethanol precipitation of 0.3M, and ethanol cleans, after the dry air, with containing 3 * SSC, and the RNA of the resuspended mark of hybridization solution 15 μ l of 0.2%SDS and 15% formaldehyde.

Slide hybridization: hybridize in the hybridizing box of the mixed instrument BIOMIXER-(CapitalBio Corp.) that tilts in three stages that placed, and in LIFTERSLIP-(Erie; Portsmouth carries out under NH), so that continue to provide hybridization solution, thereby makes hybridization more even in whole surface of glass slide, has avoided fringing effect.The hybridization of in 42 ℃ of water-baths, spending the night.Then with twice of array continuous wash: for the first time at 0.2%SDS, 42 ℃ were cleaned 5 minutes in the scavenging solution of 2 * SSC, and room temperature was cleaned 5 minutes in the scavenging solution of 0.2%SSC for the second time.With confocal scanning instrument LUXSCAN-array is scanned then, the picture that obtains is with LUXSCAN3.0-software analysis (all deriving from CapitalBio Corp.).

Data analysis: for all samples, each miRNA repeats a little to deduct the average background value.Naming a person for a particular job of expression signal＜1500 is filtered.Signal all uses the median method calibration.(Significance Analysis of Microarrays can find stat.stanford.edu/～tibs/SAM/index.html.) distinguish to the miRNA of differential expression on the World Wide Web by SAM.The significant miRNA that enough distinguishes the minimum quantity of cancer and cancer beside organism obtains by PCA (Principal Component Analysis) and SVM (Support Vector Machine) methods analyst.Predict that the most significant miRNA target obtains by four disclosed software analysis: miRBase (network address: microrna.sanger.ac.uk/sequences/), MIRANDA (available on the World Wide Web at microrna.org/), TARGETSCAN (network address: targetscan.org/), and PICTAR (network address: pictar.bio.nyu.edu/).So that reduce false positive, have only at least by three software predictions to target calculate.Patient's survivorship curve uses the method assessment of Kaplan-Meier.The acting in conjunction of concomitant variable uses CoxProportional Hazard Regression Model to detect.

Quantitative polyase chain reaction is analyzed: in order to verify the miRNA express spectra, we use the method (qRT-PCR) of quantitative polyase chain reaction with special miRNA primer total cell RNA to be analyzed.Total RNA extracts, reverse transcription, and the operation of PCR is as previously mentioned.

Case 2---miRNA expresses malignant tissue and near the healthy tissues that can distinguish in the lung cancer

Express malignant tissue and near the healthy tissues that whether can distinguish in lung cancer, squamous cell carcinoma and the gland cancer in order to study miRNA, we have used 116 pairs of primary lung cancers (60 routine squamous cell carcinomas, 43 routine gland cancer, 13 routine small cell lung cancers), 60 pairs of squamous cell carcinomas and 43 pairs of gland cancer samples are studied, these samples all have corresponding contiguous normal lung tissue, at least apart from 5 centimetres in tumour.Tissue is used liquid nitrogen flash freezer earlier, then in-80 ℃ of preservations at least 5 years.116 pairs of primary lung cancers, 60 pairs of squamous cell carcinomas and 43 pairs of gland cancer samples are divided into training group (66 pairs of primary lung cancers, 30 pairs of squamous cell carcinomas and 23 pairs of gland cancer) and check group (50 pairs of primary lung cancers, 30 pairs of squamous cell carcinomas and 20 pairs of gland cancer) at random.The point of expression signal＜1500 is filtered, and by the data of SAM analyzing and training group, equals 0 with the q value, changes conditional filtering to 29 miRNA more than or equal to 2 times.In order to set up a sorter, we have used the PCA-SVM strategy, and one group of miRNA (hsa-miR-486-5p, hsa-miR-210, hsa-miR-30a of highest score have been obtained to obtain, hsa-miR-140-3p, and hsa-miR-182) these accuracys of analyzing in initial training group are as follows: to lung cancer 98.2%, to squamous cell carcinoma 93.3%, to gland cancer 97.8% (Figures 1A, 1C, and 1E).In these five miRNA, three miRNA (hsa-miR-210, hsa-miR-30a, and hsa-miR-182) raise in tumour, and two miRNA (hsa-miR-486-5p and hsa-miR-140-3p) reduce (Table 1).After setting up sorter, the check group is used to the assessment strategy model.Accuracy in the check group is as follows: to lung cancer 92%, to squamous cell carcinoma 96.7%, to gland cancer 90% (Figures 1B, 1D, and 1F).Generally speaking, the result shows that sorter can utilize and few effectively the malignant tissue of lung cancer, squamous cell carcinoma and gland cancer is separated with healthy tissues to 5 significant miRNA.

Case 3---miRNA expresses the different pathological hypotype of not distinguishing lung cancer

Research purpose is to see that miRNA expresses three kinds of pathology hypotypes whether can distinguishing lung cancer: squamous cell carcinoma, gland cancer and small cell lung cancer, in malignant tissue and near healthy tissues.Based on the signal of miRNA in the ratio of other healthy tissues (N) miRNA of cancerous tissue (C) and cancer and the cancerous tissue, analyze the miRNA that selects differential expression in each tumour by SAM.Based on C: the miRNA of 68 differential expressions is arranged in the N ratio, three kinds of hypotypes,, the miRNA of 70 differential expressions is arranged based on the signal of miRNA in the cancerous tissue.According to the miRNA of differential expression, to 60 routine squamous cell carcinomas, 43 routine gland cancer and 13 routine small cell lung cancer samples do not have the supervision cluster analysis.Though the small cell lung cancer sample tends to poly-to same group, three kinds of hypotypes of lung cancer can't clearly be divided into three groups (Figure 2) according to their miRNA express spectra in this research.Importantly, the miRNA express spectra and the gland cancer of this analysis revealed squamous cell carcinoma do not have evident difference.On the contrary, except a few sample, the miRNA express spectra and the nonsmall-cell lung cancer of small cell lung cancer have difference.

Case 4---miRNA expresses and is associated with the pathology and the Clinical symptoms of lung cancer

Can react the miRNA feature of the different lung cancer hypotype of clinical pathology for the data that detect microarray, we have done further research.These researchs are included in the relatively expression of miRNA between many groups of groups, whether comprise smoking, age groups, and sex, the pathology classification, the differentiation classification, the TNM classification, in the tumour stage, lymphoglandula stage and the classification of tumour stage are shown in Table 2.What data analysis was used is the SAM system, based on the miRNA signal of the miRNA ratio of C: N or cancerous tissue.There are several miRNA (Table 3) to be picked out by this dual mode simultaneously.It should be noted that three miRNA (hsa-miR-205, hsa-miR-203, and hsa-miR-18b) are in low-level expression always with respect to gland cancer in squamous cell carcinoma.Two miRNA (hsa-miR-205andhsa-miR-181a) are variant expression between sex in squamous cell carcinoma.Two miRNA (hsa-miR-205and hsa-miR-31) are variant expression between high level of differentiation, medium level of differentiation, low level of differentiation in squamous cell carcinoma.A miRNA (hsa-miR-205) in nonsmall-cell lung cancer at smoking index＜400 annual and 〉=variant between the patient in 400 every year.Four clinical pathology indexs (histology hypotype, sex, differentiation and smoking index) do not have significant statistical correlations.Ironically, the lung cancer clinical pathology index that hsa-miR-205 is different with four significantly is associated.In these group data, there is not special miRNA to be associated with TNM or individual tumors stage.

Case 5---hsa-miR-31 expresses the relation with the squamous cell carcinoma prognosis

We have also studied the relation between miRNA express spectra and the patient's survival.Between squamous cell carcinoma and corresponding normal adjacent tissue, 37 miRNA differential expressions are arranged, between gland cancer and corresponding normal adjacent tissue, 22 miRNA differential expressions are arranged, between nonsmall-cell lung cancer and corresponding normal adjacent tissue, 29 miRNA differential expressions are arranged.The miRNA of all these differential expressions is used for the Kaplan-Meier survival analysis.The miRNA C of the 103 routine nonsmall-cell lung cancers that 60 initial routine squamous cell carcinomas and 43 routine gland cancer and they are merged into: the intermediate value of N is as cut-point.Kaplan-Meier analyzes and shows the high C of hsa-miR-31: and the survival negative correlation of N value and squamous cell carcinoma (p=0.007, the log-rank check, training group 60 routine patient samples, Figure3A).Further analysis has confirmed that the high expression level of hsa-miR-31 is than the low survival rate more relevant (Table 2) of low expression with squamous cell carcinoma to single argument with multivariate Cox.The univariate analysis of hsa-miR-31 and clinical pathology factor (smoking index, age, sex, differentiation, TNM classification) shows that hsa-miR-31 expression level (p=0.011) and TNM (p=0.013) have prognosis significance (Table 4) in training group.Subsequently, use the multivariable Cox proportional hazards regression models analysis of clinical pathology and branching factor to show that the hsa-miR-31 high expression level is and the incoherent significant negative prognostic factor (p=0.021 of other clinical pathology factors; Risk-ratio 3.05; 95% fiducial interval [CI], 1.187-7.838), and TNM does not have significantly related (p=0.179) (Table 5) with patient's adverse consequences.We have further studied the relation between miRNA and gland cancer and nonsmall-cell lung cancer patient's survival rate, and the result does not observe and the relevant miRNA of surviving.

In order to study the relation between hsa-miR-31 and squamous cell carcinoma patient prognosis, analyze with 20 routine squamous cell cancerous tissues independently.QRT-PCR has analyzed the expression level of miRNA.Kaplan-Meier survival analysis (Figure 3) has confirmed patient's survival rate of hsa-miR-31 high expression level (p=0.001 that significantly descends; The log-rank check, 20 pairs of tissue of patient of training group).Single argument (p=0.007) and the analysis of multivariate (p=0.029) Cox proportional hazards regression models have shown that also the hsa-miR-31 high expression level is an independently indication index (Table5) of squamous cell carcinoma negative prognosis.

This file comprises numerous concrete cases, but these do not limit to the scope of this patent application, but as the feature description under this invention particular case.Some features of under different situations, describing in this file also can applied in any combination in some situations.Otherwise, the different characteristics of under a certain situation, describing also can be separately or the part applied in any combination in multiple situation.In addition, though above features may be the formal descriptions to be used in combination, even be such declaration at first, but one or more features of being used in combination also can be cut from combination in some cases and may be comprised also from, the combination of description that subgroup is closed or the subgroup that changes is closed.

Herein one of specifically described situation a few.These situations and other situations can change and increase based on the description and the elaboration of presents.

Claims (46)

1. method of diagnosing individuality to suffer from lung cancer comprises:

A) detect the level of at least one miRNA in the individual lung tissue sample that canceration arranged under a cloud,

B) compare with reference level to the level of a miRNA of major general,

C), be to have suffered from lung cancer or had the tendency of suffering from lung cancer to classify then to individuality if having at least a miRNA level to have characteristic to change in the sample.

2. method according to claim 1, it is characterized in that: lung cancer refers to small cell lung cancer, adenocarcinoma of lung or squamous cell lung carcinoma.

3. method according to claim 1 is characterized in that: at least one miRNA refers to hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p, or corresponding autoploid.

4. method according to claim 1, it is characterized in that: comprise to the level of three miRNA of major general's individuality and comparing with reference level, wherein at least three miRNA are selected from hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p, or their corresponding autoploids.

5. method according to claim 1, it is characterized in that: comprise hsa-miR-210, hsa-miR-30a, hsa-miR-182, the expression level of hsa-miR-486-5p and hsa-miR-140-3p detects, and, if sample shows hsa-miR-210, hsa-miR-30a and hsa-miR-182 expression level increase, and hsa-miR-486-5p and hsa-miR-140-3p expression level reduce simultaneously, are to have suffered from lung cancer or had the tendency of suffering from lung cancer to classify to individuality then.

6. method according to claim 1 is characterized in that: the miRNA level uses microarray analysis to detect.

7. method according to claim 6 is characterized in that: the miRNA level is decided by the hybridization signal of miRNA on the microarray.

8. method according to claim 6 is characterized in that: the miRNA level is decided by the ratio of the signal of the hybridization signal of miRNA on the microarray and reference sample.

9. method according to claim 1 is characterized in that: the miRNA level is used Northern blot, in situ hybridization or the quantitatively method detection of reverse transcriptase polymerase chain reaction (qRT-PCR).

10. method according to claim 1 is characterized in that: the miRNA level is decided by the lymphoglandula sample, blood, and serum or lung tissue are wiped away the miRNA level in the sample.

11. distinguishing individuality suffers from a method of lung cancer and may further comprise the steps: the situation of analyzing at least one miRNA gene in this individual lung tissue sample, wherein lung tissue is to suspect the tissue that canceration is arranged, if sample shows this gene and the control sample contrast has characteristic to change, be to have suffered from lung cancer or had the tendency of suffering from lung cancer to classify then to individuality.

12. method according to claim 11 is characterized in that: the change of gene status is decided by the disappearance or the amplification of miRNA gene.

13. method according to claim 11 is characterized in that: the change of gene status is decided by the variation of miRNA gene copy number.

14. the system of lung cancer detection comprises at least one pair of primer or numerous probe, every pair of primer or each a probe miRNA in can test sample wherein, wherein at least 50% primer and probe can detect following miRNA:hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p and their autoploid.

15. using system detection of lung cancer, system comprises at least one pair of primer or numerous probe, every pair of primer or each a probe miRNA in can test sample wherein, or its corresponding miRNA gene status, wherein at least 50% primer and probe can detect following miRNA:hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p and their autoploid, wherein the horizontal occurrence characteristics variation of at least one miRNA or its autoploid is all indicating lung cancer.

At least use a pair of primer or numerous probe 16. make the lung cancer detection system, wherein every pair of primer or each probe can test sample in a miRNA, at least 50% primer or probe can detect following miRNA:hsa-miR-210, hsa-miR-30a, hsa-miR-182, hsa-miR-486-5p, hsa-miR-140-3p and their autoploid.

17. the method that patients with lung cancer is classified comprises the situation of at least one miRNA level in the individual cancerous lung tissue sample of detecting or corresponding gene, the situation with miRNA level or corresponding gene serves as to classify to patients with lung cancer in the basis then.

18. method according to claim 17 is characterized in that: comprising the miRNA that detects among Table 1 or the Table 2 or the level of corresponding autoploid, is the level of differentiation of the individual lung cancer of basis decision then with the miRNA level.

19. the method for squamous cell lung carcinoma survival patient prognosis comprises:

A) level of at least one miRNA in the individual lung tissue sample of detection,

B) level of miRNA is compared with Limiting Level,

C) the direct or negative incidence of the level of miRNA and Limiting Level is the individual prognosis of surviving.

20. method according to claim 19 is characterized in that: at least one miRNA is hsa-miR-31 or its autoploid.

21. for the survive method of individual prognosis of squamous cell lung carcinoma comprises the situation of analyzing at least one miRNA gene in the individual squamous cell lung carcinoma sample, the situation of this miRNA gene in lung cancer sample and control sample compared, and the probability to the individuality survival is height or hangs down and make assessment then.

22. method according to claim 21 is characterized in that: at least one miRNA gene is gene or its autoploid of coding hsa-miR-31.

23., it is characterized in that: comprise that further decision is fit to individual treatment plan according to claim 21 or 22 described methods.

24. according to the arbitrary described method of claim 21-23, it is characterized in that: the miRNA level is passed through Northernblot, in situ hybridization, or quantitatively reverse transcriptase polymerase chain reaction (qRT-PCR) detects.

25. according to the arbitrary described method of claim 21-23, it is characterized in that: the miRNA level is decided by the lymphoglandula sample, blood, and serum or lung tissue are wiped away the miRNA level in the sample.

26. use one or more pairs of primers or probe to be squamous cell lung carcinoma patient prognosis, wherein one or more pairs of primers or the probe miRNA in can test sample, the level of miRNA and the ratio of Limiting Level and individual survival probability positive correlation or negative correlation.

27. method according to claim 26 is characterized in that: at least one miRNA is hsa-miR-31 or its autoploid.

28. use one or more pairs of primers or probe to be fabricated to the reagent or the system of squamous cell lung carcinoma patient prognosis, wherein one or more pairs of primers or the probe miRNA in can test sample, the level of miRNA and the ratio of Limiting Level and individual survival probability positive correlation or negative correlation.

29. method according to claim 28 is characterized in that: at least one miRNA is hsa-miR-31 or its autoploid.

30. for improving the method for patients with lung cancer survival state, comprise the reagent that can reduce a miRNA level that gives individual effective dose, wherein the ratio of the level of miRNA and Limiting Level and individual survival probability negative correlation.

31. method according to claim 30 is characterized in that: at least one miRNA is hsa-miR-31 or its autoploid.

32. method according to claim 30 is characterized in that: at least one miRNA is hsa-miR-210, hsa-miR-30a, hsa-miR-182 or their autoploid.

33. method according to claim 30 is characterized in that: reagent wherein is sense-rna.

34. method according to claim 30 is characterized in that: reagent wherein is siRNA.

35. method according to claim 30, it is characterized in that: comprise one or more reagent that can reduce at least two miRNA levels that give individual effective dose, wherein these two miRNA are selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182 or their autoploid.

36. method according to claim 30, it is characterized in that: comprise one or more reagent that can reduce at least three miRNA levels that give individual effective dose, wherein these three miRNA are selected from hsa-miR-31, hsa-miR-210, hsa-miR-30a, hsa-miR-182 or their autoploid.

37. method according to claim 30 is characterized in that: comprise give individual effective dose can reduce hsa-miR-31, hsa-miR-210, hsa-miR-30a, one or more reagent of the level of hsa-miR-182 or their autoploid.

38. use can reduce the medicine that the reagent manufacturing of miRNA level is used for improving the patients with lung cancer survival state, wherein the ratio of the level of miRNA and Limiting Level and individual survival state negative correlation.

39. according to the described method of claim 38, it is characterized in that: at least one miRNA is hsa-miR-31 or its autoploid.

40. according to the described method of claim 38, it is characterized in that: at least one miRNA is hsa-miR-210, hsa-miR-30a, hsa-miR-182 or their autoploid.

41. according to the described method of claim 38, it is characterized in that: reagent wherein is sense-rna.

42. according to the described method of claim 38, it is characterized in that: reagent wherein is siRNA.

43. medicinal composition comprises acceptable carrier and the reagent that reduces the miRNA expression level on the pharmacopedics, wherein at least one miRNA is hsa-miR-31 or its autoploid.

44. medicinal composition comprises acceptable carrier and the reagent that reduces the miRNA expression level on the pharmacopedics, wherein at least one miRNA is hsa-miR-210, hsa-miR-30a, hsa-miR-182 or their autoploid.

45. according to claim 43 or 44 described medicinal compositions, it is characterized in that: reagent is sense-rna.

46. according to claim 43 or 44 described medicinal compositions, it is characterized in that: reagent is siRNA.

Images