CN102770561A

CN102770561A - Tissue-based micro-RNA methods for diagnosis of different subtypes of lung cancer

Info

Publication number: CN102770561A
Application number: CN2010800647948A
Authority: CN
Inventors: 吴莹; 卢韶华; 黄威; 朱虹光; 李健
Original assignee: Fudan University
Current assignee: SHANGHAI LABWAY CLINICAL LABORATORY Co.,Ltd.
Priority date: 2009-12-24
Filing date: 2010-12-24
Publication date: 2012-11-07
Anticipated expiration: 2030-12-24
Also published as: CN102770561B

Abstract

The invention provides diagnostic kits comprising a plurality of nucleic acid molecules encoding microRNA sequences for identifying one or more mammalian target cells exhibiting lung cancer, wherein the nucleic acid molecules are differentially expressed in target cells and in control cells. The invention further provides methods for identifying one or more mammalian target cells exhibiting lung cancer by using said nucleic acid molecules, and methods and pharmaceutical compositions for preventing or treating lung cancer.

Description

Be used to diagnose the little-RNA method based on tissue of different subtype lung cancer

Invention field

The present invention relates to the miRNA biomarker and corresponding method through confirming, to be used for the lung cancer of different subtype in orthopaedic surgical operations operation reliably and the examination of living tissue lung tissue tissue, especially for the lung cancer of difference different subtype.Said lung cancer hypotype comprises gland cancer lung cancer, prognosis of squamous cell lung cancer and small cell lung cancer.

Background of invention

Lung cancer still is modal reason in the masculinity and femininity cancer associated death in the world.The 1400000 newly-increased cases of having an appointment in 2009 according to estimates; Follow average 2.51% annual growth (Frost&Sullivan estimation); These patient overwhelming majority that were diagnosed as lung cancer 15 in 2009 will die from this disease (Higgins; M.J.et al. (2009) Expert Rev Anticancer Ther 9,1365-1378).Although in the past decades Chinese and foreign department technology and combination therapy certain improvement has been arranged, at US and European difference total 5 annual survival rates only about 15% of patients with lung cancer by stages.

Lung cancer be categorized as small cell lung cancer (small cell lung cancer, SCLC) or nonsmall-cell lung cancer (non-small cell lung cancer, NSCLC).It is that NSCLC comprises gland cancer and squamous cell shape cell carcinoma that main (80%) cancerous lung tissue is learned type.Smoking is the most important risks and assumptions that lung cancer takes place, and accounts for 50% among 80% and the female lung cancer patient in the male lung cancer case in the world wide.

Lung cancer therapy is according to different cancer hypotype and different.It is surgical operation that the treatment of early stage NSCLC is selected, and total 5 annual survival rates of 40% are arranged.But most of patients the time has been in late period in diagnosis, makes first-line treatment only be confined to the multiple medicines combined chemotherapy and the expection survival rate is less than 8 months.Latest Development aspect targeted therapy requires nonsmall-cell lung cancer (NSCLC) 30 hypotype classification more accurately.The cancer angiogenesis inhibitor can make squamous cell shape cell carcinoma patient be easier to take place abnormal response (Lebanoy, D. (2009) J Clin Oncol 27,2030-2037).Small cell lung cancer (SCLC) patient is the highest hypotype of lethality in the lung cancer, and mortality is higher than 90%.Although in the early metaphase patient, often observe high initial reaction rate, the meta survival rate was also had an appointment 20 months.But surgical operation almost is invalid to the treatment of SCLC, and chemotherapy or chemotherapy radiotherapy are united and be only treatment and select.

Except the difference treatment to different subtype and different cause of disease lung cancer, special, standardized detection has also limited the ability of instantly patient being implemented the right treatment of abundant tactful chemical combination to the difference between human observer with shortage.Treatment decision-making to patients with lung cancer is individual very soon will be based on detailed cancer and host's characteristic.The specific molecular biomarker that is used to distinguish the lung cancer hypotype is absolute demand.

Many diagnostic assays also are subject to the analysis that generally is based on single molecular indexes, and this analysis possibly influence the confidence level and/or the accuracy of detection.In addition, single index can not be carried out detailed forecasts to latency stage, cancer progress etc. usually.Therefore, still need excavate alternative molecular indexes and detection mode and overcome these limitations.

One of solution to this problem can be based on control RNA molecule; Particularly based on the non-coding RNA by the endogenous expression of the evolution conservative of 20-25 nucleic acid size, such RNA can mediate that said target mrna is expressed and since it was found, be proved microRNA (miRNA) (Bartel, D.P. (2004) Cell 116 of participation cell development, differentiation, propagation and apoptosis with this before 10 years; 281-297; Ambros, V. (2004) Nature 431,350-355; He, L.et al. (2004) Nat Rev Genet 5,522-531).And because it is in external highly stable and in vivo long-term existence, miRNA has the unexistent advantage of other mRNA (Lu, J.et al., (2005) Nature 435, the 834-838 as the cancer biomarker; Lim, L.P.et al., (2005) Nature 433,769-773).

MiRNA results from former precursor (pre-miRNA) of transcribing and being processed into by RNase III Drosha stem-loop structure.After the transporte to cells matter, the ring that the RNase III of another Dicer by name shears the pre-miRNA hair clip forms short dsrna, and wherein a chain is integrated into miRNA-protein complex (miRNP) with ripe miRNA.MiRNA guiding miRNP wherein is to said target mrna performance function (Bartel, D.P. (2004) Cell 23,281-292; He, L.and Hannon, G.J. (2004) Nat Rev Genet 5,522-531).

According to the complementary degree between miRNA and its target, miRNA can guide different regulation processes.With the high complementary mRNA of miRNA through disturbing (RNAi) identical mechanism to be sheared by specificity with RNA.Therefore, in this case, miRNA is as short interfering rna (siRNA) performance function.The mRNA that target and miRNA complementarity are more weak or be directed to the cell degradation approach or suppress not influencing under the mRNA horizontal state by translation.But it is still disputable how miRNA suppresses the translating mechanism of said target mrna.

High path miRNA quantitative technique is the TaqMan miRNA detection on basis like the miRNA chip, with the real-time RT-PCR, has been proved to be the strong instrument that the full oncogene of research is organized whole miRNA spectrum.More and more data shows that the unusual adjusting of miRNA expression is relevant with the generation and/or the progress of particular type cancer.For example: two miRNA, miR-15 and miR-16-1 are proved the genetic locus that is located at disappearance in the lymphocytic leukemia (CLL), and the disappearance or the downward modulation of miRNA gene are arranged in 70% CLL patient.And the downward modulation of miR-15 and miR-16-1 also has discovery in knot rectum knurl becomes, and the expression of miRNA let-7 is everlasting and is reduced (Michael, M.Z.et al. (2003) Mol Cancer Res 1,882-891 in the lung cancer; Mayr, C.et al. (2007) Science 315,1576-1579).In fact, but change with miRNA according to the cancer dependency that miRNA expresses and often to be positioned at heredity district supposition miRNA suppressor oncogene and oncogene (Esquela-Kerscher, A.and Slack, F.J (2006) Nat Rev Cancer 6, the 259-269 relevant with cancer; Calin, G.A.and Croce, C.M. (2007) J Clin Invest 117,2059-2066; Blenkiron, C.and Miska, E.A. (2007) Hum Mol Genet 16, R106-R113).The miRNA expression pattern has highlighted it and can be used to diagnose the potential with the prognosis biomarker in existing people's cancer of describing.

Some research report miRNA express spectra (Johnson, S.M.et al. (2005) Cell 120,635-647 in lung cancer; Liang, Y.et al. (2008) BMC Med Genomics 1,61; Kumar, M.S.et al. (2008) Proc Natl Acad Sci USA 105,3903-3908; Miko, E.et al. (2009) Exp Lung Res 35,646-664; Xie, Y et al. (2009) Lung Cancer May 13; Lebanony, D.et al. (2009) J Clin Oncol 27,2030-2037; Kauppinen, S.et al. (2009) Clin Cancer Res 15,1177-1183; Mascaux, C.et al. (2009) Eur Respir J33,352-359).Identical ground, these researchs show that all specific miRNA compares with non-malign lung tissue unconventionality expression is arranged in malignant tissue.And research shows that some miRNA maybe relevant (Yu, S.L.et al. (2008) Cancer Cell 13,48-57 with prognosis; Raponi, M.et al (2009) Cancer Res69,5776-5783).Therefore, these miRNA can be to giving a clue with vicious transformation and the relevant cell processes of progress.

Thus; Still the diagnosis index that needs (a series of), especially with " expression characteristic (signature) " or " molecular imprinting " form can carry out fast, reliable and save and spend the property evaluation and/or treatment shows as the cell that maybe might develop into dissimilar lung cancer.In addition, the method that still need can identify and treat the target cell of cancer phenotype accordingly simultaneously

Goal of the invention and summary of the invention

The purpose of this invention is to provide the miRNA biomarker and corresponding method through confirming, to be used for the lung cancer of orthopaedic surgical operations operation reliably and examination of living tissue lung tissue different subtype, especially for the lung cancer of difference different subtype.Specifically; Through confirming multiple nucleic acid molecule; Lung cancer shows as gland cancer lung cancer, squamous cell carcinoma and small cell lung cancer; Every kind of nucleic acid molecule encoding miRNA sequence, and in the said multiple nucleic acid molecule one or more by in the target cell of analyzing with in control cells, compare differential expression, and the nucleic acid molecule of one or more differential expressions can be used as the expression of nucleic acid biomarker that a kind of indication lung cancer exists together.

More specifically, target of the present invention provides effective miRNA biomarker, with Mammals target cell and/or difference gland cancer lung cancer, prognosis of squamous cell lung cancer or the small cell lung cancer that is used to identify that one or more shows lung cancer.

In addition, the present invention also aims to provide corresponding method, to be used to identifying that one or more shows the lung cancer and the prevention of the Mammals target cell of lung cancer, difference different subtype or treats this type of disease.

These and other purpose will become clear from following description, and its theme through independent claim is reached.Certain preferred embodiments of the present invention then limits through the theme of dependent claims.

In first aspect; The present invention relates to be used to identify the diagnostic kit of the molecule marker of one or more Mammals target cells that show gland cancer lung cancer; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents a kind of expression of nucleic acid biomarker together, this biomarker is the indication that gland cancer lung cancer exists.

The expression of nucleic acid biomarker that this paper limits can comprise at least four kinds of nucleic acid molecule.

In concrete embodiment, said expression of nucleic acid biomarker comprises the nucleic acid molecule of at least a coding miRNA sequence, and it is expressed in one or more target cells to compare with one or more normal control cells and is raised; And the nucleic acid molecule that comprises at least a coding microrna sequences, it is expressed in one or more target cells to compare with one or more normal control cells and is reduced.

In preferred embodiments, said expression of nucleic acid biomarker comprises any one or the multiple nucleic acid molecule of coding hsa-miR-27a, hsa-miR-29a, hsa-miR-34a and hsa-miR-375..

Particularly preferably; Compare with said one or more normal control cells, the expression of any one or the multiple nucleic acid molecule of hsa-miR-34a and hsa-miR-375 up-regulated and coding hsa-miR-27a and hsa-miR-29a is reduced in said one or more target cells.

In more preferred, the expression of nucleic acid biomarker comprises arbitrary or a plurality of nucleic acid combination among coding hsa-miR-34a/hsa-miR-27a and the hsa-miR-34a/hsa-miR-29a.Especially the expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-29a, hsa-miR-27a and hsa-miR-34a.

Second aspect; The present invention relates to be used to identify the diagnostic kit of the molecule marker of one or more Mammals target cells that show prognosis of squamous cell lung cancer; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents a kind of expression of nucleic acid biomarker together, this biomarker is the indication that prognosis of squamous cell lung cancer exists.

Expression of nucleic acid biomarker defined herein can comprise at least four kinds of nucleic acid molecule.

In preferred embodiments, the expression of nucleic acid biomarker comprises the nucleic acid molecule of at least a coding miRNA sequence, and it is expressed in one or more target cells to compare with one or more normal control cells and is raised; And the nucleic acid molecule that comprises at least a coding miRNA sequence, it is expressed in one or more target cells to compare with one or more normal control cells and is reduced.

In a more preferred embodiment, the expression of nucleic acid biomarker comprises any one or the multiple nucleic acid molecule of coding hsa-miR-205, hsa-miR-25, hsa-miR-29a and hsa-miR-375.

Especially preferredly be, compare that hsa-miR-205 and hsa-miR-25 are raised in said one or more target cells, and the expression of the nucleic acid molecule of coding hsa-miR-29a and hsa-miR-375 is reduced with said one or more normal control cells.

In a more preferred embodiment; The expression of nucleic acid biomarker comprises coding hsa-miR-205/hsa-miR-29a; Hsa-miR-25/hsa-miR-29a, any one of hsa-miR-205/hsa-miR-375 and hsa-miR-25/hsa-miR-375 or the combination of multiple nucleic acid.Especially, the expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-205, hsa-miR-25 and hsa-miR-29a.

In the third aspect; The present invention relates to be used to identify the diagnostic kit of the molecule marker of one or more Mammals target cells that show small cell lung cancer; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents a kind of expression of nucleic acid biomarker together, this biomarker is the indication that small cell lung cancer exists.

Expression of nucleic acid biomarker defined herein can comprise at least five kinds of nucleic acid molecule.

In more preferred, the expression of nucleic acid biomarker comprises any one or the multiple nucleic acid molecule of coding hsa-miR-25, hsa-miR-27a, hsa-miR-29a, hsa-miR-29b and hsa-miR-375.

Especially preferredly be; Compare with said one or more normal control cells; Hsa-miR-25 and hsa-miR-375 are raised in said one or more target cells, and the expression of the nucleic acid molecule of coding hsa-miR-27a, hsa-miR-29a and hsa-miR-29b is reduced.

In a more preferred embodiment, the expression of nucleic acid biomarker comprises any one or the combination of multiple nucleic acid of coding hsa-miR-25/hsa-miR-27a, hsa-miR-375/hsa-miR-27a, hsa-miR-25/hsa-miR-29a and hsa-miR-375/hsa-miR-29a.Especially, the expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-29a and hsa-miR-375.

Fourth aspect; The present invention relates to be used for the diagnostic kit of molecule marker that prognosis of squamous cell lung cancer and gland cancer lung cancer are differentiated; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents a kind of expression of nucleic acid biomarker together, this biomarker is the indication that prognosis of squamous cell lung cancer or gland cancer lung cancer exist.

In preferred embodiments, the expression of nucleic acid biomarker comprises the nucleic acid molecule of at least a coding miRNA sequence, and it is expressed in one or more target cells to compare with one or more control cells and is raised; And the nucleic acid molecule that comprises at least a coding miRNA sequence, it is expressed in one or more target cells to compare with one or more control cells and is reduced.

In a more preferred embodiment, the expression of nucleic acid biomarker comprises any one or the multiple nucleic acid molecule of coding hsa-miR-205, hsa-miR-25, hsa-miR-27a and hsa-miR-375.

Especially preferredly be, compare that hsa-miR-205, hsa-miR-25, hsa-miR-27a are raised in said one or more target cells, and the expression of the nucleic acid molecule of coding hsa-miR-375 is reduced with said one or more control cells.

In a more preferred embodiment, expression of nucleic acid biomarker any one or the multiple nucleic acid that comprise coding hsa-miR-205/hsa-miR-375 and hsa-miR-25/hsa-miR-375 makes up.Especially, the expression of nucleic acid biomarker comprises a kind of nucleic acid molecule combination of coding hsa-miR-205, hsa-miR-25 and hsa-miR-375.

The 5th aspect; The present invention relates to be used for the diagnostic kit of molecule marker that small cell lung cancer and gland cancer lung cancer are differentiated; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents a kind of expression of nucleic acid biomarker together, this biomarker is the indication that small cell lung cancer or gland cancer lung cancer exist.

Expression of nucleic acid biomarker defined herein can comprise at least six kinds of nucleic acid molecule.

In a more preferred embodiment, the expression of nucleic acid biomarker comprises any one or the multiple nucleic acid molecule of coding hsa-miR-25, hsa-miR-27a, hsa-miR-29a, hsa-miR-29b, hsa-miR-34a and hsa-miR-375.

Especially preferredly be; Compare with said one or more control cells; Hsa-miR-25 and hsa-miR-375 are raised in said one or more target cells, and the expression of the nucleic acid molecule of coding hsa-miR-27a, hsa-miR-29a, hsa-miR-29b and hsa-miR-34a is reduced.

In a more preferred embodiment, the expression of nucleic acid biomarker comprises any one or the combination of multiple nucleic acid of coding hsa-miR-25/hsa-miR-27a, hsa-miR-375/hsa-miR-27a, hsa-miR-25/hsa-miR-29a and hsa-miR-375/hsa-miR-29a.Especially, the expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-25, hsa-miR-34a and hsa-miR-375.

The 6th aspect; The present invention relates to be used for the diagnostic kit of molecule marker that small cell lung cancer and prognosis of squamous cell lung cancer are differentiated; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents a kind of expression of nucleic acid biomarker together, this biomarker is the indication that small cell lung cancer or prognosis of squamous cell lung cancer exist.

In preferred said scheme, the expression of nucleic acid biomarker comprises any one or the multiple nucleic acid molecule of coding hsa-miR-205, hsa-miR-27a, hsa-miR-29a, hsa-miR-29b, hsa-miR-34a and hsa-miR-375.

Especially preferredly be; Compare with said one or more control cells; Hsa-miR-375 is raised in said one or more target cells, and the expression of the nucleic acid molecule of coding hsa-miR-205, hsa-miR-27a, hsa-miR-29a, hsa-miR-29b and hsa-miR-34a is reduced.

In a more preferred embodiment, expression of nucleic acid biomarker any one or the multiple nucleic acid that comprise coding hsa-miR-375/hsa-miR-27a and hsa-miR-375/hsa-miR-29a makes up.Especially, the expression of nucleic acid biomarker comprises coding hsa-miR-29b and the combination of hsa-miR-375 nucleic acid.

The 7th aspect the present invention relates to be used to identify the method for one or more Mammals target cells that show gland cancer lung cancer, prognosis of squamous cell lung cancer or small cell lung cancer, and said method comprises:

(a) collection patient's examination of living tissue or surgical operation tissue;

(b) on slide glass, prepare tissue slice;

(c) the nucleic acid molecule biomarker with at least a coding microrna sequences hybridizes in the section on the slide glass;

(d) perhaps the expression of miRNA is carried out quantitatively at microscopically through digital pathology scheme;

(e) in described one or more target cells, confirm multiple nucleic acid molecule expression level, every kind of nucleic acid molecule microrna sequences of all encoding;

(f) expression level of definite said multiple nucleic acid molecule in one or more control cells; And

(g) through contrasting in step (e) and the expression level separately that is obtained (f); From said multiple nucleic acid molecule, identify one or more nucleic acid molecule of differential expression in target cell and control cells; The nucleic acid molecule of wherein said one or more differential expressions is represented like expression of nucleic acid biomarker defined herein together, and it is the indication that gland cancer lung cancer, prognosis of squamous cell lung cancer or small cell lung cancer exist.

Especially preferred is that this method realizes with in situ hybridization.

Quantitative definite in order to carry out, the miRNA biomarker of 7 empirical tests of use: hsa-miR-205, hsa-miR-25, hsa-miR-27a, hsa-miR-29a, hsa-miR-29b, hsa-miR-34a and hsa-miR-375.

For expression level stdn, can preferably use the coding hsa-miR-24 expression of nucleic acid molecule of stably express in lung tissue with the nucleic acid molecule biomarker of the coding microrna sequences that is obtained.Negative control as the nucleic acid molecule biomarker expression level of the coding miRNA sequence that is obtained can preferably use the coding hsa-miR-122 expression of nucleic acid molecule of in lung tissue, not expressing.

Eight aspect the present invention relates to prevent or treat the method for lung cancer, and this method comprises: (a) through using method as herein described in one or more target cells, to identify the expression of nucleic acid biomarker; And (b) in said one or more cells, change the expression of one or more nucleic acid molecule of the coding microrna sequences that said expression of nucleic acid biomarker comprised; Said change is carried out in such a way; Be that its expression that is expressed in the nucleic acid molecule that is raised in said one or more target cells is reduced, and its expression that is expressed in the nucleic acid molecule of being reduced in said one or more target cells is raised.

The 9th aspect; The present invention relates to be used to prevent and/or treat the lung cancer drugs compsn; Said compsn comprises one or more nucleic acid molecule; Every kind of equal encoding sequence of nucleic acid molecule; Part is complementary at least for said sequence and the coded microrna sequences of the nucleic acid molecule that its expression is raised in said one or more target cells defined herein, and/or said sequence is corresponding to the coded microrna sequences of nucleic acid molecule that its expression is reduced in said one or more target cells defined herein.

At last, the tenth aspect the present invention relates to aforementioned pharmaceutical compositions is used for preventing and/or treating lung cancer drugs in preparation purposes.

Other embodiments of the present invention will be illustrated in detailed description subsequently.

The accompanying drawing summary

Fig. 1: having described in brief summary of the invention the 7th aspect according to detected representation is the schema that the present invention of single or many target cells of gland cancer lung cancer, prognosis of squamous cell lung cancer or small cell lung cancer is used for confirming the basic method steps of a certain expression biomarker.Especially use in-situ hybridization method to distinguish different lung cancer hypotypes.

Fig. 2 A: explain that according to being used for detected representation be that the present invention of single or many target cells of gland cancer lung cancer belongs to the especially people miRNA of preferred expression biomarker in first aspect.Also show simultaneously expression level and the accuracy (that is: raise or reduce) of comparing these miRNA in the gland cancer patients with lung cancer with normal lung tissue.Data show that the gland cancer lung cancer in the FFPE surgical operation tissue can distinguish with normal lung tissue reliably.

Fig. 2 B: progressively described and be used for detecting one or more target cell that FFPE surgical operation tissue shows gland cancer lung cancer in the first aspect present invention to organizing the logistic regression analysis of (hsa-miR-27a, hsa-miR-29a and hsa-miR-34a).Data show that the gland cancer lung cancer in the FFPE surgical operation tissue can distinguish with normal lung tissue (AUC=0.925) reliably.

Fig. 3 A: explain that according to being used for detected representation be that the present invention of single or many target cells of prognosis of squamous cell lung cancer belongs to the especially people miRNA of preferred expression biomarker in second aspect.Also show expression level and the accuracy (that is: raise or reduce) of comparing these miRNA in the gland cancer patients with lung cancer with normal lung tissue.Data show that the prognosis of squamous cell lung cancer in the FFPE surgical operation tissue can distinguish with normal lung tissue reliably.

Fig. 3 B: progressively described in the second aspect according to being used for detecting the present invention of single or many target cells that FFPE surgical operation tissue shows as prognosis of squamous cell lung cancer to the logistic regression analysis of one (hsa-miR-205, hsa-miR-25 and the hsa-miR-29a) that make up.Data show that the gland cancer lung cancer in the FFPE surgical operation tissue can distinguish with normal lung tissue (AUC=0.961) reliably.

Fig. 4 A: explain that according to being used for detected representation be that the present invention of single or many target cells of small cell lung cancer belongs to the especially people miRNA of preferred expression biomarker in the third aspect.Also show expression level and the accuracy (that is: raise or reduce) of comparing these miRNA among the small cell lung cancer patient with normal lung tissue.Data show that the small cell lung cancer in the FFPE surgical operation tissue can distinguish with normal lung tissue reliably.

Fig. 4 B: progressively described in the third aspect according to being used for detecting the present invention of single or many target cells that FFPE surgical operation tissue shows as small cell lung cancer to the logistic regression analysis of one (hsa-miR-29a and the hsa-miR-375) that make up.Data show that the small cell lung cancer in the FFPE surgical operation tissue can distinguish with normal lung tissue (AUC=0.998) reliably.

Fig. 5 A: explain according to being used for differentiating that the present invention of prognosis of squamous cell lung cancer and gland cancer lung cancer belongs to the especially people miRNA of preferred expression biomarker in fourth aspect.Also show expression level and the accuracy (that is: raise or reduce) of comparing these miRNA among the prognosis of squamous cell lung cancer patient with the gland cancer patients with lung cancer.Data show that the prognosis of squamous cell lung cancer in the FFPE surgical operation tissue can distinguish with gland cancer lung cancer reliably.

Fig. 5 B: progressively described in the fourth aspect according to be used for differentiating FFPE surgical operation tissue prognosis of squamous cell lung cancer can with the present invention of gland cancer lung cancer logistic regression analysis to one (hsa-miR-205, hsa-miR-25 and the hsa-miR-375) that make up.Data show that prognosis of squamous cell lung cancer can be distinguished with gland cancer lung cancer (AUC=0.922) reliably in the FFPE surgical operation tissue.

Fig. 6 A: explain according to being used for differentiating that the present invention of small cell lung cancer and gland cancer lung cancer belongs to the especially people miRNA of preferred expression biomarker aspect the 5th.Also show expression level and the accuracy (that is: raise or reduce) of comparing these miRNA among the small cell lung cancer patient with the gland cancer patients with lung cancer.Data show that the small cell lung cancer in the FFPE surgical operation tissue can distinguish with gland cancer lung cancer reliably.

Fig. 6 B: progressively described in the 5th aspect according to be used for differentiating FFPE surgical operation tissue small cell lung cancer can with the present invention of gland cancer lung cancer logistic regression analysis to one (hsa-miR-25, hsa-miR-34a and the hsa-miR-375) that make up.Data show that small cell lung cancer can be distinguished with gland cancer lung cancer (AUC=0.991) reliably in the FFPE surgical operation tissue.

Fig. 7 A: explain according to being used for differentiating that the present invention of small cell lung cancer and prognosis of squamous cell lung cancer belongs to the especially people miRNA of preferred expression biomarker aspect the 6th.Also show expression level and the accuracy (that is: raise or reduce) of comparing these miRNA among the small cell lung cancer patient with the prognosis of squamous cell lung cancer patient.Data show that the small cell lung cancer in the FFPE surgical operation tissue can distinguish with prognosis of squamous cell lung cancer reliably.

Fig. 7 B: progressively described in the 6th aspect according to be used for differentiating FFPE surgical operation tissue small cell lung cancer can with the present invention of prognosis of squamous cell lung cancer logistic regression analysis to one (hsa-miR-29b and the hsa-miR-375) that make up.Data show that small cell lung cancer can be distinguished with prognosis of squamous cell lung cancer (AUC=0.982) reliably in the FFPE surgical operation tissue.

Detailed Description Of The Invention

The present invention is based on lung cancer and can diagnoses reliably and hypotype is distinguished according to hypersensitivity and specific specific miRNA express spectra, and confirm at this these express biomarkers and generally include the discovery outside this expection aspect the upper and lower accent two.More particularly, can realize the early diagnosis and the hypotype of lung cancer are distinguished through analyzing whole miRNA expression pattern and/or independent miRNA expression level.The present invention of following illustration can implement under the condition that does not have not concrete in this article any one or more element that discloses, one or more restrictions suitably.

The present invention will describe according to specific embodiment and with reference to accompanying drawing, but the present invention is not limited, and limited by claims.Described accompanying drawing only is schematically, is considered to nonrestrictive.

When term " comprises " when being used in specification sheets of the present invention and claims, it does not get rid of other element or step.Be the object of the invention, term " by ... form " be considered to the preferred embodiment that term " comprises ".If group is restricted to and comprises at least one fixed number purpose embodiment hereinafter, this also has been understood that to disclose the group of preferably only being made up of these embodiments.

Use indefinite article or definite article for example " one " or " a kind of " when referring to the singulative noun, when " said ", comprise the plural form of this noun, unless otherwise indicated.

Term " approximately " is meant that in the present invention the particularity that it will be apparent to those skilled in the art that the technique effect that still can guarantee the purpose characteristic is interval.This term ordinary representation departs from indicator value ± 10%, preferred ± 5%.

In addition, term first, second, third, (a) and (b), (c) etc. are used to distinguish similar elements in specification sheets and claims, are not that description order or chronological order are necessary.The term that should understand application like this is interchangeable under appropriate, and the embodiment that the present invention describes can be to be different from other sequential operation of described herein or illustration.

Term further be defined in following using a technical term the time provide.

Following term or definition have been merely to be understood the present invention and provides.These definition should not be considered to have less than the scope that it will be apparent to those skilled in the art that.

Term used herein " cancer " (being also referred to as " cancer (carcinoma) ") is often referred to the malignant growth of any kind, promptly compares any morphology and/or the physiology change (based on hereditary reprogrammed (geneticre-programming)) that show or have the target cell that the cancer tendency takes place with unaffected (health) wild-type control cells.The example of this change can relate to cell size and shape (become greatly or diminish), cell proliferation (cell count increase), cytodifferentiation (physiology change of state), apoptosis (apoptosis) or cell survival.

Term used herein " lung cancer " refers to uncontrolled cell growth in the lung tissue, or the cancerous growths of lung tissue.

Comprise gland cancer lung cancer, prognosis of squamous cell lung cancer and small cell lung cancer at this use a technical term " different subtype of lung cancer ".

" gland cancer lung cancer " or " gland property lung cancer " is one type of nonsmall-cell lung cancer.80% lung cancer is nonsmall-cell lung cancer (NSCLC), and wherein 50% is gland cancer.Gland cancer lung cancer betides the peripheral region of lung, can before diagnosis, exist for a long time.Be modal lung cancer type among the women, also common in the non-smoker.

" prognosis of squamous cell lung cancer " is one type of nonsmall-cell lung cancer.About 30%NSCLC is a prognosis of squamous cell lung cancer.Prognosis of squamous cell lung cancer often betides the segmental bronchus (big airways) of lung central part.Much human promptly has symptom in early days, and normal is spitting of blood (bringing up blood).

" small cell lung cancer " (SCLC) is considered to originate from the neuroendocrine cell of component part segmental bronchus (air flue) epithelium (lining attaches).SCLC accounts for 18% of all lung cancer cases.SCLC is had an aggressive very much.Growth is diffused into liver, lung, bone and brain very soon and through blood flow.When diagnosis, be easy in these organs, find the metastases kitchen range.

Refer under a cloudly at least suffer from lung cancer at this used term " patient ", or the people of certain type of lung cancer; The healthy subjects of term " healthy individuals " or this cancer phenotype of " normal control " Chang Zhiwu.But, in some applications, for example when more dissimilar lung cancer, have the individuality of other types lung cancer often to be considered to " contrast ".

The sample that in vitro method of the present invention, is used to detect should be collected with clinical acceptable manner usually, preferably collects with protection nucleic acid (particularly RNA) or proteinic mode.Sample to be analyzed typically is a tissue.In addition, blood and other type sample also can use.

Term used herein " microRNA " (or " miRNA ") is that its its ordinary meaning in this area (is summarized referring to for example Bartel D.P. (2004) Cell 23,281-292; He, L.and Hannon, G.J. (2004) Nat.Rev.Genet.5,522-531).Therefore, " microRNA " is meant the RNA molecule derived from the genomic gene seat, and it is from forming the transcript processing of partial rna precursor miRNA structure.Ripe miRNA normal length is 20,21,22,23,24 or 25 Nucleotide, and the Nucleotide of other number also can exist, for example 18,19,26 or 27 Nucleotide.

The miRNA encoding sequence has and flanking gene group sequence paired potentiality; Ripe miRNA is placed within the non-complete paired RNA duplex (this paper is also referred to as stem-ring or hairpin structure or pre-miRNA), and said duplex is as the midbody that carries out miRNA processing from longer precursor transcript.This processing typically continuous action of two species specific endonucleases through being called Drosha and Dicer respectively takes place.Drosha produces the miRNA precursor (this paper is also referred to as " pre-miRNA ") that typically is folded into hair clip or stem-ring structure from primary transcript (this paper is also referred to as " pri-miRNA ").From this miRNA precursor, through Dicer cutting miRNA duplex, its one arm at hair clip or stem-ring structure comprises ripe miRNA, comprises the sections (being commonly referred to miRNA*) of similar size at other one arm.MiRNA is directed to its said target mrna then bringing into play its function, and miRNA* is degraded.In addition, miRNA typically derived from the prediction protein coding region different gene group sections.

Term used herein " miRNA precursor " (or " precursor miRNA " or " pre-miRNA ") is meant the part of processing the miRNA primary transcript of ripe miRNA from it.Typically, pre-miRNA is folded into stable hair clip (being duplex) or stem-ring structure.Hairpin structure typically length is a 50-80 Nucleotide, preferred 60-70 Nucleotide (counting miRNA residue, with miRNA paired residue, and any sections that interleaves, but get rid of the more sequence of far-end).

Term used herein " nucleic acid molecule of coding microrna sequences " is meant any nucleic acid molecule of coding microRNA (miRNA).Therefore, this term not only refers to ripe miRNA, also refers to corresponding aforesaid precursor miRNA and elementary miRNA transcript.In addition, the invention is not restricted to the RNA molecule, also comprise the dna molecular of respective coding microRNA, the dna molecular that for example produces through rt miRNA sequence.The nucleic acid molecule of microrna sequences of the present invention of the encoding single miRNA sequence (being individual miRNA) of typically encoding.But, the also possible two or more miRNA sequences of this nucleic acid molecule encoding (being two or more miRNA), for example a transcription unit is included in two or more miRNA sequences of regulating under sequence such as promotor or the transcription terminator control commonly used.

It (is the nucleotide sequence (coupling of 5' → 3') or (be nucleic acid array complementation in the coded miRNA (sequence of 5' → 3') or in other words mate the reverse complementary sequence (3' → 5') molecule) of coded miRNA sequence corresponding to coded miRNA (5' → 3') molecule of sequence) and " antisense nucleic acid molecule " that the term used herein nucleic acid molecule of microrna sequences " coding " also is understood to include " the phosphorothioate odn molecule is arranged ".Term used herein " complementation " is meant that " antisense " sequence of nucleic acid molecules and corresponding " justice is arranged " sequence of nucleic acid molecules (having the sequence that is complementary to antisense sequences) form the ability of base pair, preferred Watson-Crick base pair.

Within the scope of the present invention, two nucleic acid molecule (promptly " justice being arranged " and " antisense " molecule) can be complementary fully, and promptly they do not contain any base mispairing and/or Nucleotide extra or disappearance.Perhaps, two molecules comprise one or more base mispairing or different on their Nucleotide sum (causing owing to add or lack).Preferably, " complementation " nucleic acid molecule comprises with the sequence that is included in corresponding " justice is arranged " nucleic acid molecule and shows at least 10 complementary fully continuous nucleotides.

Therefore, the multiple nucleic acid molecule that is included in the coding miRNA sequence in the diagnostic kit of the present invention can comprise that one or more " have the phosphorothioate odn molecule " and/or one or more " antisense nucleic acid molecules ".Sometimes; Diagnostic kit comprises one or more " has the phosphorothioate odn molecule " (being miRNA sequence itself); Said molecule has been considered to form all or at least inferior set of the miRNA (being molecule marker) of differential expression; The miRNA of said differential expression is the indication that has or take place the particular condition tendency, and this paper is lung cancer.On the other hand; When diagnostic kit comprises one or more " antisense nucleic acid molecules " (promptly with miRNA sequence complementary sequence), said molecule can comprise be suitable for detecting and/or quantitative given sample in the probe molecule (being used to carry out hybridization assays) and/or the Oligonucleolide primers (for example being used for rt or PCR uses) of one or more specific (complementations) miRNA sequence.

The multiple nucleic acid molecule of definition can comprise at least 2 kinds, at least 10 kinds, at least 50 kinds, at least 100 kinds, at least 200 kinds, at least 500 kinds, at least 1000 kinds, at least 10000 kinds or at least 100000 kinds of nucleic acid molecule, every kind of molecule encoding miRNA sequence in the present invention.

Term used herein " differential expression " is meant that the expression level of specific miRNA in target cell changes than normal healthy controls cell or other diseases type sample, and it can be to raise (promptly miRNA concentration increases in target cell) or downward modulation (promptly miRNA concentration reduces or disappears in target cell).In other words, nucleic acid molecule is activated in target cell to than level higher or lower in control cells.

Within the scope of the present invention; Nucleic acid molecule is considered to differential expression; If the corresponding expression level of this nucleic acid molecule in target cell and control cells typically differs at least 5% or at least 10%, preferably at least 20% or at least 25%, most preferably at least 30% or at least 50%.Therefore, the latter's value raises at least 1.3 times or at least 1.5 times corresponding to the expression level of given nucleic acid molecule in target cell respectively than the wild-type control cells, otherwise perhaps the expression level in target cell is reduced at least 0.7 times or at least 0.5 times.

Term used herein " expression level " is meant the degree that specific miRNA sequence is transcribed from its genomic gene seat, promptly miRNA at one or more by the concentration in the analysis of cells.

As stated, term " control cells " typically is meant and is collected in (health) individual cells sample with lung cancer phenotypic characteristic.But in some applications, for example when relatively showing dissimilar lung cancer, the cell that derives from other types lung cancer typically is considered to " control cells ".

The definite of expression level typically follows the standard program of having set up well known in the art (referring to for example Sambrook; J.et al. (1989) Molecular Cloning:A Laboratory Manual.2nd Ed.; Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY; Ausubel, F.M.et al. (2001) Current Protocols in Molecular Biology.Wiley&Sons, Hoboken, NJ).Confirm and to carry out at rna level, for example use the miRNA specific probe to carry out the Northern engram analysis, perhaps behind rt (and clone) RNA crowd, for example carry out at dna level through quantitative PCR or real time pcr.Any nucleic acid molecule of the above-mentioned miRNA sequence of analysis of encoding " confirmed " to comprise in term used herein.But,, typically only measure the concentration of ripe miRNA owing to pri-miRNA and re-mRNA half life weak point.

In concrete embodiment, the standard value of the expression level that in some independent measurements (for example two, three, five or ten measurements) of given sample and/or the some measurements in a group target cell or control cells, obtains is used to analyze.Standard value can use any method known in the art to obtain.For example, the scope of MV ± 2SD (standard deviation) or MV ± 3SD can be used as standard value.

Difference between one or more target cells that obtained and the expression level of one or more control cells can stdn (or normalization method; Normalize) to the further contrast nucleic acid expression level of house-keeping gene for example, the expression level of house-keeping gene is known not according to the morbid state of cell and difference.House-keeping gene for example comprises beta-actin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein P1 etc.In preferred embodiments, contrast nucleic acid is the another kind of miRNA of stably express in known different non-cancer and cancer (preceding) state at cell.

But, replace in any experiment, confirming the expression level of one or more control cells, also can be based on experimental evidence and/or prior art D.D. one or more cutoff value to specific cells phenotype (being morbid state).In this case, the corresponding expression level of one or more target cells can be confirmed with the contrast miRNA that is used for standardized stably express.If the expression level of " stdn " of calculating is higher than the cutoff value of corresponding definition, then this discovery is the indication that genetic expression is raised.Otherwise if the expression level of " stdn " of calculating is lower than the cutoff value of corresponding definition, then this discovery is the indication of down regulation of gene expression.

In the present invention, term " diagnosing and/or distinguish different lung cancer hypotypes " also refers to prediction and probability analysis (meaning " diagnosis ").Biomarker described here and method are intended to be used for and comprise therapeutic intervention, Case definition such as the staging clinical decision of disease surveillance and the treatment pattern aspect of keeping watch on.According to the present invention, the stage casing result to an object condition detection can be provided.This stage casing result can diagnose the individuality of suffering from this disease together with the auxiliary doctor of other information, nurse or other practitioners.In addition, this invention can be used as the pair cell sample and carry out carcinous change detection, and to the doctor the favourable information in order to diagnosis is provided.Once more, the present invention can be used for the differential diagnosis of different lung cancer hypotypes.

In the present invention, the nucleic acid molecule of one or more differential expressions of being identified is represented a kind of expression of nucleic acid biomarker together, and this expression of nucleic acid characteristic is the indication that in target cell, has lung cancer.Term used herein " expression biomarker " is meant one group of nucleic acid molecule (for example miRNA), and wherein the expression level of each nucleic acid molecule is different between the cell in patients with lung cancer source and normal control cell.Among this paper, the expression of nucleic acid biomarker also refers to a group echo and represents minimum purpose (difference) nucleic acid molecule that every kind of nucleic acid molecule encoding can be identified the miRNA sequence of the phenotype state of an individuality.

In a more preferred embodiment, expression of nucleic acid characteristic (signature) comprises the nucleic acid molecule of one or more coding hsa-miR-27a (SEQ ID NO:3), hsa-miR-29a (SEQ ID NO:4), hsa-miR-34a (SEQ ID NO:6) and hsa-miR-375 (SEQ ID NO:7).

Especially preferredly be; Compare with one or more control cells; Hsa-miR-34a in one or more target cells (SEQ ID NO:6) and hsa-miR-375 (SEQ ID NO:7) are raised, and encode (SEQ ID NO:3) and the expression of any one or the multiple nucleic acid molecule of hsa-miR-29a (SEQ ID NO:4) is reduced.

In a more preferred embodiment, the expression of nucleic acid biomarker nucleic acid that comprises any one or multiple coding hsa-miR-34a (SEQ ID NO:6)/hsa-miR-27a (SEQ ID NO:3) and hsa-miR-34a (SEQ ID NO:6)/hsa-miR-29a (SEQ ID NO:4) makes up.Especially the expression of nucleic acid biomarker comprises a kind of nucleic acid molecule combination of coding hsa-miR-29a (SEQ ID NO:4), hsa-miR-27a (SEQ ID NO:3) and hsa-miR-34a (SEQ ID NO:6).

The nucleic acid molecule biomarker of the coding miRNA sequence that obtains for stdn can preferably use coding hsa-miR-24 (SEQ ID NO:8) the expression of nucleic acid molecule of stably express in lung tissue.Negative control as the nucleic acid molecule biomarker expression level of the coding miRNA sequence that obtains can preferably use coding hsa-miR-122 (SEQ ID NO:9) the expression of nucleic acid molecule of in lung tissue, not expressing.

The above-mentioned miRNA nucleotide sequence that relates to is listed in table 1.

Table 1

miRNA	Sequence (5' → 3')
		Biomarker
hsa-miR-27a	uucacaguggcuaaguuccgc
		hsa-miR-29a	uagcaccaucugaaaucgguua
hsa-miR-34a	uggcagugucuuagcugguugu
		hsa-miR-375	uuuguucguucggcucgcguga
Contrast
		hsa-miR-24	uggcucaguucagcaggaacag
hsa-miR-122	uggagugugacaaugguguuug

All miRNA sequences disclosed herein all have been kept at (http://microrna.sanger.ac.uk/ in the miRBase DB; Also referring to Griffiths-Jones S.et al. (2008) Nucl.Acids Res.36, D154-D158).

Can be any subgroup of nucleic acid molecule multiform property at the term " at least a nucleic acid " of this use; That is: arbitrary, wantonly two, wantonly three, wantonly four, wantonly five, wantonly six, wantonly seven, wantonly eight, wantonly nine, wantonly ten by that analogy nucleic acid molecule, each nucleic acid molecule encoding one is contained in the miRNA sequence at this expression of nucleic acid biomarker of confirming.

In second aspect; The present invention relates to be used to identify the diagnostic kit of the molecule marker of one or more Mammals target cell that shows prognosis of squamous cell lung cancer; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents the expression of nucleic acid biomarker together, this biomarker is the indication that has prognosis of squamous cell lung cancer.

In a more preferred embodiment, the expression of nucleic acid biomarker comprises the nucleic acid molecule of one or more coding hsa-miR-205 (SEQ ID NO:1), hsa-miR-25 (SEQ ID NO:2), hsa-miR-29a (SEQ ID NO:4) and hsa-miR-375 (SEQ ID NO:7).

Especially preferredly be; Compare with one or more control cells, hsa-miR-205 in one or more target cells (SEQ ID NO:1), hsa-miR-25 (SEQ ID NO:2) are raised and the expression of any one or the multiple nucleic acid molecule of hsa-miR-29a that encodes (SEQ ID NO:4) and hsa-miR-375 (SEQ ID NO:7) is reduced.

In preferred version further, the expression of nucleic acid biomarker comprises the nucleic acid combination of one or more coding hsa-miR-205 (SEQ ID NO:1)/hsa-miR-29a (SEQ ID NO:4), hsa-miR-25 (SEQ ID NO:2)/hsa-miR-29a (SEQ ID NO:4), hsa-miR-205 (SEQ ID NO:1)/hsa-miR-375 (SEQ ID NO:7), hsa-miR-25 (SEQ ID NO:2)/hsa-miR-375 (SEQ ID NO:7) arbitrarily.Especially the expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-205 (SEQ ID NO:1), hsa-miR-25 (SEQ ID NO:2) and hsa-miR-29a (SEQ ID NO:4).

The above-mentioned miRNA nucleotide sequence that relates to is listed in table 2.

Table 2

miRNA	Sequence (5' → 3')
		Biomarker
hsa-miR-205	uccuucauuccaccggagucug
		hsa-miR-25	cauugcacuugucucggucuga
hsa-miR-29a	uagcaccaucugaaaucgguua
		hsa-miR-375	uuuguucguucggcucgcguga
Contrast
		hsa-miR-24	uggcucaguucagcaggaacag
hsa-miR-122	uggagugugacaaugguguuug

All miRNA sequences in this announcement all have been kept at (http://microrna.sanger.ac.uk/ in the miRBase DB; Also referring to Griffiths-Jones S.et al. (2008) Nucl.Acids Res.36, D154-D158).

In the third aspect; The present invention relates to be used to identify the diagnostic kit of the molecule marker of one or more Mammals target cell that shows small cell lung cancer; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents a kind of expression of nucleic acid biomarker together, this biomarker is the indication that has small cell lung cancer.

The expression of nucleic acid biomarker that this paper limits can comprise at least five kinds of nucleic acid molecule.

In a further preferred embodiment, the expression of nucleic acid biomarker comprises the nucleic acid molecule of one or more coding hsa-miR-25 (SEQ ID NO:2), hsa-miR-27a (SEQ ID NO:3), hsa-miR-29a (SEQ ID NO:4), hsa-miR-29b (SEQ ID NO:5) and hsa-miR-375 (SEQ ID NO:7).

Especially preferredly be; Compare with one or more control cells; In one or more target cells; Hsa-miR-25 (SEQ ID NO:2) and hsa-miR-375 (SEQ ID NO:7) are raised, and the expression of any one or more nucleic acid molecule of coding hsa-miR-27a (SEQ ID NO:3), hsa-miR-29a (SEQ ID NO:4), hsa-miR-29b (SEQ ID NO:5) is reduced.

In embodiment preferred further, the expression of nucleic acid biomarker comprises arbitrarily that the nucleic acid of one or more coding hsa-miR-25 (SEQ ID NO:2)/hsa-miR-27a (SEQ ID NO:3), hsa-miR-375 (SEQ ID NO:7)/hsa-miR-27a (SEQ ID NO:3), hsa-miR-25 (SEQ ID NO:2)/hsa-miR-29a (SEQ ID NO:4) and hsa-miR-375 (SEQ ID NO:7)/hsa-miR-29a (SEQ ID NO:4) makes up.Especially the expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-29a (SEQ ID NO:4) and hsa-miR-375 (SEQ ID NO:7).

The above-mentioned miRNA nucleotide sequence that relates to is listed in table 3.

Table 3

miRNA	Sequence (5' → 3')
		Biomarker
hsa-miR-25	cauugcacuugucucggucuga
		hsa-miR-27a	uucacaguggcuaaguuccgc
hsa-miR-29a	uagcaccaucugaaaucgguua
		hsa-miR-29b	uagcaccauuugaaaucaguguu
hsa-miR-375	uuuguucguucggcucgcguga
		Contrast
hsa-miR-24	uggcucaguucagcaggaacag
		hsa-miR-122	uggagugugacaaugguguuug

In fourth aspect; The present invention relates to be used for the diagnostic kit of differential diagnosis prognosis of squamous cell lung cancer and gland cancer lung cancer; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents the expression of nucleic acid biomarker together, this biomarker is the indication that has prognosis of squamous cell lung cancer or gland cancer lung cancer.

In a further preferred embodiment, the expression of nucleic acid biomarker comprises the nucleic acid molecule of one or more coding hsa-miR-205 (SEQ ID NO:1), hsa-miR-25 (SEQ ID NO:2), hsa-miR-27a (SEQ ID NO:3) and hsa-miR-375 (SEQ ID NO:7).

Especially preferredly be; Compare with one or more control cells; In one or more target cells; Hsa-miR-205 (SEQ ID NO:1), hsa-miR-25 (SEQ ID NO:2), hsa-miR-27a (SEQ ID NO:3) are raised, and the expression of any one or more nucleic acid molecule of coding hsa-miR-375 (SEQ ID NO:7) is reduced.

In embodiment preferred further, the expression of nucleic acid biomarker comprises arbitrarily that the nucleic acid of one or more coding hsa-miR-205 (SEQ ID NO:1)/hsa-miR-375 (SEQ ID NO:7) and hsa-miR-25 (SEQ ID NO:2)/hsa-miR-375 (SEQ ID NO:7) makes up.Especially the expression of nucleic acid biomarker comprises a kind of nucleic acid molecule combination of coding hsa-miR-205 (SEQ ID NO:1), hsa-miR-25 (SEQ ID NO:2) and hsa-miR-375 (SEQ ID NO:7).

The above-mentioned miRNA nucleotide sequence that relates to is listed in table 4.

Table 4

miRNA	Sequence (5' → 3')
		Biomarker
hsa-miR-205	uccuucauuccaccggagucug
		hsa-miR-25	cauugcacuugucucggucuga
hsa-miR-27a	uucacaguggcuaaguuccgc
		hsa-miR-375	uuuguucguucggcucgcguga
Contrast
		hsa-miR-24	uggcucaguucagcaggaacag
hsa-miR-122	uggagugugacaaugguguuug

Aspect the 5th; The present invention relates to be used for the diagnostic kit of differential diagnosis small cell lung cancer and gland cancer lung cancer; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents the expression of nucleic acid biomarker together, this biomarker is the indication that has small cell lung cancer or gland cancer lung cancer.

The expression of nucleic acid biomarker that this paper limits can comprise at least six kinds of nucleic acid molecule.

In a further preferred embodiment, the expression of nucleic acid characteristic comprises the nucleic acid molecule of one or more coding hsa-miR-25 (SEQ ID NO:2), hsa-miR-27a (SEQ ID NO:3), hsa-miR-29a (SEQ ID NO:4), hsa-miR-29b (SEQ ID NO:5), hsa-miR-34a (SEQ ID NO:6) and hsa-miR-375 (SEQ ID NO:7).

Especially preferredly be; Compare with one or more control cells; In one or more target cells, hsa-miR-25 (SEQ ID NO:2) and hsa-miR-375 (SEQ ID NO:7) are raised, and coding hsa-miR-27a (SEQ ID NO:3); Hsa-miR-29a (SEQ ID NO:4), the expression of any one or more nucleic acid molecule of hsa-miR-29b (SEQ ID NO:5) and hsa-miR-34a (SEQ ID NO:6) is reduced.

In embodiment preferred further, the expression of nucleic acid biomarker comprises arbitrarily that the nucleic acid of one or more coding hsa-miR-25 (SEQ ID NO:2)/hsa-miR-27a (SEQ ID NO:3), hsa-miR-375 (SEQ ID NO:7)/hsa-miR-27a (SEQ ID NO:3), hsa-miR-25 (SEQ ID NO:2)/hsa-miR-29a (SEQ ID NO:4) and hsa-miR-375 (SEQ ID NO:7)/hsa-miR-29a (SEQ ID NO:4) makes up.Especially the expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-25 (SEQ ID NO:2), hsa-miR-34a (SEQ ID NO:6) and hsa-miR-375 (SEQ ID NO:7).

The above-mentioned miRNA nucleotide sequence that relates to is listed in table 5.

Table 5

miRNA	Sequence (5' → 3')
		Biomarker
hsa-miR-25	cauugcacuugucucggucuga
		hsa-miR-27a	uucacaguggcuaaguuccgc
hsa-miR-29a	uagcaccaucugaaaucgguua
		hsa-miR-29b	uagcaccauuugaaaucaguguu
hsa-miR-34a	uggcagugucuuagcugguugu
		hsa-miR-375	uuuguucguucggcucgcguga
Contrast
		hsa-miR-24	uggcucaguucagcaggaacag
hsa-miR-122	uggagugugacaaugguguuug

Aspect the 6th; The present invention relates to be used for the diagnostic kit of differential diagnosis small cell lung cancer and prognosis of squamous cell lung cancer; Said test kit comprises multiple nucleic acid molecule; Every kind of nucleic acid molecule encoding microrna sequences; One or more of wherein said multiple nucleic acid molecule target cell and in one or more control cells differential expression, and the nucleic acid molecule of wherein said one or more differential expressions represents a kind of expression of nucleic acid biomarker together, this biomarker is the indication that has small cell lung cancer or prognosis of squamous cell lung cancer.

In a further preferred embodiment, the expression of nucleic acid characteristic comprises the nucleic acid molecule of one or more coding hsa-miR-205 (SEQ ID NO:1), hsa-miR-27a (SEQ ID NO:3), hsa-miR-29a (SEQ ID NO:4), hsa-miR-29b (SEQ ID NO:5), hsa-miR-34a (SEQ ID NO:6) and hsa-miR-375 (SEQ ID NO:7).

Especially preferredly be; Compare with one or more control cells; In one or more target cells; Hsa-miR-375 (SEQ ID NO:7) is raised, and the expression of any one or more nucleic acid molecule of coding hsa-miR-205 (SEQ ID NO:1), hsa-miR-27a (SEQ ID NO:3), hsa-miR-29a (SEQ ID NO:4), hsa-miR-29b (SEQ ID NO:5) and hsa-miR-34a (SEQ ID NO:6) is reduced.

In embodiment preferred further, the expression of nucleic acid biomarker comprises the nucleic acid combination of one or more coding hsa-miR-375 (SEQ ID NO:7)/hsa-miR-27a (SEQ ID NO:3), hsa-miR-375 (SEQ ID NO:7)/hsa-miR-29a (SEQ ID NO:4) arbitrarily.Especially, the expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-29b (SEQ ID NO:5) and hsa-miR-375 (SEQ ID NO:7).

The above-mentioned miRNA nucleotide sequence that relates to is listed in table 6.

Table 6

?miRNA	Sequence (5' → 3')
		Biomarker
?hsa-miR-205	uccuucauuccaccggagucug

hsa-miR-27a	uucacaguggcuaaguuccgc
		hsa-miR-29a	uagcaccaucugaaaucgguua
hsa-miR-29b	uagcaccauuugaaaucaguguu
		hsa-miR-34a	uggcagugucuuagcugguugu
hsa-miR-375	uuuguucguucggcucgcguga
		Contrast
hsa-miR-24	uggcucaguucagcaggaacag
		hsa-miR-122	uggagugugacaaugguguuug

All miRNA sequences that this paper discloses all have been kept at (http://microrna.sanger.ac.uk/ in the miRBase DB; See also Griffiths-Jones S.et al. (2008) Nucl.Acids Res.36, D154-D158).

(b) on slide glass, prepare tissue slice;

Especially preferred is that this method realizes with in situ hybridization.

For quantitatively determined; Use the miRNA biomarker of 7 empirical tests: hsa-miR-205 (SEQ ID NO:1); Hsa-miR-25 (SEQ ID NO:2), hsa-miR-27a (SEQ ID NO:3), hsa-miR-29a (SEQ ID NO:4); Hsa-miR-29b (SEQ ID NO:5), hsa-miR-34a (SEQ ID NO:6) and hsa-miR-375 (SEQ ID NO:7).

For the expression level that the said labeled nucleic acid molecule thing of standard code miRNA sequence obtains, can preferably use the expression of nucleic acid molecule of said coding hsa-miR-24 (SEQ ID NO:8), it is stably express in lung tissue.And the negative control of the expression level that obtains for the said labeled nucleic acid molecule thing of coding miRNA sequence can preferably use the expression of nucleic acid molecule of described coding hsa-122 (SEQ ID NO:9), and it is not expressed in lung tissue.

Hybridization in situ technique allows specific nucleic acid molecule to be detected in karyomit(e), cell or the tissue slice preserved of quilt on form.The binding immunoassay cytochemistry, in situ hybridization can be at DNA, and mRNA and protein level connect microcosmic topology information of same gene activity.Two types on-radiation hybridizing method is arranged: direct method and indirect method.Direct method is to use resorcinolphthalein or other fluorescein stains and Nucleotide directly be coupled (Baumann, J.G.J.et al. ((1980) Exp.Cell Res.138,485 – 490).Indirect method is to use digoxigenin (detecting through specific antibody) and vitamin H (streptavidin detection) (Leary, J.L et al (1983) Proc.Natl.Acad.Sci.USA 80,4045 – 4049).

As used herein; Term " nucleic acid molecule that changes coding miRNA sequence is expressed " is meant that any manipulation to the specific nucleic acid molecule changes with the expression level that causes said molecule, promptly compares the corresponding miRNA that produces different amounts with the expression of " wild-type " (being the contrast of unmodified).As used herein, term " different amount " had both comprised with the contrast of unmodified and had compared higher amount, also comprised lower amount.In other words, can be the expression (promptly particularly transcribing) of raising (promptly activating) or downward modulation (promptly suppressing) nucleic acid molecule like manipulation defined herein.

In the present invention; The expression of one or more nucleic acid molecule of the coding microrna sequences that is comprised in the expression of nucleic acid characteristic is modified by this way, and its expression that expression that is expressed in the nucleic acid molecule that is raised in said one or more target cells is reduced and it is expressed in the nucleic acid molecule of being reduced in said one or more target cells is raised thus.In other words; The modification of the expression of the specific nucleic acid molecule of coding miRNA sequence with the generation of the antimode pattern (anti-cyclical) of the regulating effect of said molecule in said one or more cancer target cells, with " overactivity " of in said one or more target cells, disturbing the molecule that is raised and/or " defective is active " of the molecule that recovers to be reduced.

In a preferred embodiment of the inventive method, the expression of downward modulation nucleic acid molecule comprises that the nucleic acid molecule of the microrna sequences complementary sequence of the nucleic acid molecule encoding that coding and quilt are reduced imports in one or more target cells.

Term as used herein " complementary sequence " is meant that " complementation " nucleic acid molecule (this paper is also referred to as " antisense nucleic acid molecule ") that imports in one or more cells can form base pair with endogenous " justice is arranged " nucleic acid molecule that raises, preferred Watson-Crick base pair.

Two kinds of nucleic acid molecule (promptly " justice being arranged " and " antisense " molecule) can be complete complementary, and promptly it does not contain any base mispairing and/or interpolation or disappearance Nucleotide.In other embodiments, these two kinds of molecules comprise one or more base mispairing or its Nucleotide sum different (because due to interpolation or disappearances).In other embodiment, " complementation " nucleic acid molecule comprise one section with " justice is arranged " nucleic acid molecule that raises at least ten continuous nucleotides of the complete complementary of sequence of comprising.

" complementation " nucleic acid molecule (i.e. the nucleic acid molecule of the microrna sequences complementary nucleotide sequence of the nucleic acid molecule encoding of coding and downward modulation) can be the DNA-or the RNA molecule of natural generation or the synthetic nucleic acid molecule that in its sequence, comprises one or more same type or one or more dissimilar modified nucleotides.

For example, possibly comprise at least one ribonucleotide main chain unit and at least one deoxyribonucleotide main chain unit by this nucleic acid molecule.In addition; It is 2'-O-methyl group or 2'-O-methoxy group (being also referred to as 2'-O-methylates) that said nucleic acid molecule can contain one or more RNA backbone modifications; It prevents at substratum amplifying nucleic acid enzyme liberating; And be that the kernel that also prevents the reticent mixture nucleicacidase of RNA inducibility separates importantly, cause the irreversible inhibition of miRNA.Another possible modification (its function equivalence methylates in 2'-O-) comprises locked nucleic acid (LNA); Representative contains the nucleic acid analog of one or more LNA nucleotide monomer; Said monomer is simulated at RNA has locking bifuran sugar unit (referring to for example Orom in the sugared conformation; U.A.et al. (2006) Gene 372,137-141).

Developed another kind of miRNA expression silencing gene recently.These chemical engineering oligonucleotide that are called " antagomirs " are RNA molecules (Krutzfeldt, J.et al. (2005) Nature 438,685 – 689) of 23 Nucleotide of strand of puting together with SUV.Another selection as this chemically modified oligonucleotide has produced the microRNA suppressor factor that can in cell, express that from transgenic, produces as RNA.These competitive inhibitors that are called " microRNA sponge (microRNA sponges) " are the transcripts of expressing from strong promoter; The a plurality of series combination site (Ebert that contains interested microRNA; M.S.et al. (2007) Nat.Methods 4,721-726).

In order to illustrate any potential association of the miRNA that differentiates in the sample before carcinous or the cancer, can carry out preparation function analysis about the discriminating of the combinable mRNA target sequence of said miRNA.Based on finding that miRNA both can participate in tumor suppressor and also can participate in tumour and take place that (F.J (2006) is like preamble for Esquela-Kerscher, A.and Slack; Calin, G.A.and Croce, C.M. (2007) is like preamble; Blenkiron, C.and Miska, E.A. (2007) is like preamble), can infer that the mRNA target site of this miRNA comprises tumor suppressor gene and oncogene.

If nucleic acid molecule comprises the sequential element that contains relevant for transcribing and/or translate adjusting information; And this sequence " operably connects " in the nucleotide sequence of coded polypeptide, claims that then this nucleic acid molecule perhaps can " make nucleotide sequence express " for " ability express nucleic acid molecule ".Operably connect is wherein said adjusting sequential element and the connection of the sequence of being expressed (and/or sequence of expression) mutually can make that the mode of genetic expression is connected.

For the definite character of the essential regulatory region of genetic expression can be different in different plant species; But these zones all comprise promotor usually; It contains two promotors in prokaryotic organism, the DNA element that promptly instructs the DNA element of transcription initiation and when being transcribed into RNA, send translation initiation signal.This promoter region generally includes the 5' non-coding region of participating in transcribing with translation initiation, as in prokaryotic organism-35/-10 box and Shine-Dalgarno element perhaps TATA box, CAAT sequence and the 5'-in eukaryotic cell add the cap element.These zones also can comprise enhanser or prevent sub-element and translation signals and leader sequence with the specific compartment of natural polypeptides target in host cell.In addition, the 3' non-coding sequence can contain the regulatory element of participating in Transcription Termination, polyadenylation etc.Yet if the function of these terminator sequences in specific host cell is unsatisfactory, the signal that can be used in performance function in this cell replaces.

In addition, the expression like the nucleic acid molecule of this paper definition also can influence (as stated) through for example there being the Nucleotide of modifying.For example; Locked nucleic acid (LNA) monomer is considered to through strengthening to the resistance of degraded and through the stable functional transformation period (Naguibneva that increases miRNA in the body for reticent active crucial miRNA-target duplex structure; I.et al. (2006) Biomed.Pharmacother.60,633 – 638).

Therefore, the nucleic acid molecule of the present invention that is imported in one or more cells that provide can comprise the adjusting sequence, preferred promoter sequence, the optional transcription termination sequence that also comprises.Said promotor can allow composing type or inducible gene expression.Suitable promotor comprises intestinal bacteria (E.coli) lacUV5 and tet (tsiklomitsin is replied) promotor, T7 promotor and SV40 promotor or CMV promotor.

Nucleic acid molecule of the present invention also can be included in carrier or other cloning vector such as plasmid, phagemid, phage, clay or the artificial chromosome.In preferred embodiments, said nucleic acid molecule is included in the carrier, particularly is included in the expression vector.Can comprise duplicating with control sequence and give the selective marker that cells transfected can be selected phenotype the nucleotide sequence of the genetic constructs that this expression vector defines except above-mentioned adjusting sequence and coding as the present invention derived from the species compatible with the host who is used to express.Many suitable carriers known in the art and commercially available such as pSUPER and pSUPERIOR

Within the scope of the present invention; Suitable pharmaceutical compositions comprises and is suitable for those compsns that oral, rectum, nose, part (comprising through containing clothes and hypogloeeis), peritonaeum and parenteral (comprising intramuscular, subcutaneous or intravenously) give, perhaps through sucking or be blown into those compsns that give.Can the part or general give.Preferred administered through oral or intravenous route give.Said preparation can be packaged as separate dosage units.

Pharmaceutical composition of the present invention comprises any pharmaceutical dosage forms that this area is confirmed; For example capsule, micro-capsule, cachet, pill, tablet, powder, pilule (pellet), many granular preparations (for example pearl, particle or crystal), aerosol, sprays, foam, solution, dispersion agent, tincture, syrup, elixir, suspension-s, water-in-oil emulsion such as ointment, and water external emulsion such as emulsion, lotion and face cream.

The preparation method who uses the acceptable composition of pharmacology and set up can be formulated as pharmaceutical composition (Gennaro with above-mentioned (" justice is arranged " and " antisense ") nucleic acid molecule; A.L.and Gennaro; A.R. (2000) Remington:The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams&Wilkins; Philadelphia, PA; Crowder, T.M.et al. (2003) A Guide to Pharmaceutical Particulate Science.Interpharm/CRC, Boca Raton, FL; Niazi, S.K. (2004) Handbook of Pharmaceutical Manufacturing Formulations, CRC Press, Boca Raton, FL).

For pharmaceutical compositions, can use the inorganic or organic excipients (being carrier) of pharmacy inert.In order to prepare for example pill, tablet, capsule or particle, can use for example lactose, talcum, Triple Pressed Stearic Acid and salt thereof, fat, wax, solid or liquid polyol, natural oil or winterized stearin.Be used to produce solution, suspension-s, milk sap, aerosol mixture or before using reprovision comprise water, alcohol, glycerine, polyvalent alcohol and suitable mixture thereof and vegetables oil as the appropriate excipients of the powder of solution or aerosol mixture.

Said pharmaceutical composition also can contain additive, like filling agent, wedding agent, moistening agent, glidant, stablizer, sanitas, emulsifying agent, reaches the material that other solvent or solubilizing agent are perhaps realized storage effect.The latter can be regarded as and can nucleic acid molecule be mixed in slowly-releasing or lasting release or the targeted system as in liposome, nano particle and the micro-capsule.

For the intravital great majority tissue of target, need clinical feasible nothing wound strategy being oriented to cell like this pharmaceutical composition of this paper definition.In the past, certain methods has obtained great treatment benefit through the siRNA with reasonable dosage in mouse and primate body are gone in intravenous injection, and does not have significantly restriction toxicity.

A kind of method comprise with passerby's chain of miRNA (miRNA* chain) and SUV or derivatives thereof/conjugate covalent coupling with the absorption of the cell surface ldl receptor that promotes to express through omnipresence (Soutschek, J.et al. (2004) Nature 432,173-178).Perhaps, the oligonucleotide (LNA-antimiR) that the locked nucleic acid of unconjugated PBS-preparation is modified can be used for general carry (Elmen, J.et al. (2008) Nature 452,896-899).The method of the another kind of miRNA of conveying comprises uses polyoxyethylene glycol to make the miRNA capsulation become the specific lipid body with the absorption that reduces scavenger cell and strengthen cycling time.These specific nucleic acid particles (stable nucleic acid-lipid granule or SNALP) are delivered to liver with miRNA effectively and (and do not arrive other organ (referring to for example Zimmermann, T.S.et al. (2006) Nature 441,111-114)).In recent years; Agent delivery (the Akinc of one type of novel lipid appearance delivery of molecules that is called lipoids (lipidoid) (based on alkyl acrylate or alkyl-acrylic amide and primary amine or the puting together addition of secondary amine and synthesize) as the RNAi therapeutical agent described; A.et al. (2008) Nat.Biotechnol.26,561-569).

Further cell-specific target strategy comprises miRNA is mixed with a kind of fusion rotein; This fusion rotein is made up of the targeting antibodies fragment that is connected with protamine; Said protamine is the basic protein (Song that makes the DNA nucleation in the sperm and pass through charge bonded miRNA; E.et al. (2005) Nat.Biotechnol.23,709-717).Recently developed multiple modification and the change that above-mentioned basic carrying method is carried out.These technology are known in the art, and summarize at for example de Fougerolles A.et al. (2007) Nat.Rev.Drug Discov.6,443-453; Kim, D.H.and Rossi, J.J. (2007) Nat.Genet.8 is among the 173-184.

The present invention further describes with following embodiment through accompanying drawing, and said accompanying drawing and embodiment are the purpose for illustration special embodiment of the present invention, should not be construed as the meaning that limits the scope of the invention by any way.

Embodiment

Embodiment 1: patient's data

In finding research, patients with lung cancer frozen tissue sample 125 examples of filing during the middle mountain 2007-2009 of hospital have been collected.All patients have informed consent to participating in scientific research.Compiling according to agreement of tissue sample by the approval of the last marine mountain comment council of association of hospital.After more than being organized in operation and exsomatizing, use the OCT embedding immediately, freezing fast with liquid nitrogen, and be stored at-80 ° of C with for use.Adjoin morphologic healthy tissues (apart from cancerous tissue 10cm at least) from identical patients with lung cancer.These samples comprise 36 routine gland cancer lung cancer, 30 routine prognosis of squamous cell lung cancer and 16 routine small cell lung cancers and the corresponding healthy tissues of 44 examples.

In checking research, collected from the cancerous lung tissue of 215 examples of filing during the last marine mountain 2004-2008 of hospital through formalin fixed paraffin embedding (FFPE).Comprise 54 routine gland cancer lung cancer in these FFPE tissues, 50 routine prognosis of squamous cell lung cancer and 56 routine small cell lung cancers and the corresponding healthy tissues of 55 examples.Finding and verifying that the foundation characteristic of the tumor specimen in the research sees table-7 for details.

Table 7: the characteristic of tissue sample in finding research and checking research

Tissue sample	The discovery group	The checking group
			Normal lung tissue	44	55
Lung cancer
			Gland cancer	36	54
The squama cancer	30	50
			Small cell lung cancer	16	56
Sum	126	215

Patient data (age, sex, image data, treat-ment, other medical conditions, family history or the like) is used to mate collected different samples from hospital database.Pathology follow (histologic analysis that for example carries out through h and E (H&E) dyeing) is used for clearly confirming the morbid state (for example normal control group, gland cancer lung cancer, prognosis of squamous cell lung cancer or small cell lung cancer) of given sample and the consistent classification that guarantees sample.

Embodiment 2: specimen preparation

In finding research, carry out the laser capture micro-dissections with specific isolation tumor cell group (about 200000 cells) to each cancer sample is optional.In brief, transparent transfer film is applied to the surface of tissue slice or sample.At microscopically, observe the thin tissue section that places on the slide glass, and the identification of cell crowd is to separate.When the cell of selecting is positioned at the center of field of view, activate the integrated Optics in Microscope device of near-infrared laser diode.Pulse laser beam activates the spot (spot) on the transfer film, makes the cytogamy of this film and following selection.The transfer film that will have the bonded cell is then peeled off from said thin tissue is cut into slices and (is summarized referring to for example Emmert-Buck M.R.et al. (1996) .Science 274,998-1001; Espina, V.et al. (2007) Expert Rev.Mol.Diagn.7,647-657).Basically instruct the preparation freezing microtome section and use laser capture microscope (Arcturus VeritasTM Laser Capture Microdissection Instrument (Molecular Devices like manufacturer; Inc.; Sunnyvale, CA USA) catches step.

For the transformation that helps to study clinical implementation from exploratory, the FFPE surgical tissue is used for checking research.Research and analyse in case the FFPE tissue is selected to, the HE section will be prepared for checking the shared ratio of tumor section in each sample.Surpass 75% neoplastic cell if a tumor group is woven with, it will be considered to be suitable for analyze and need not do further tumour cell purifying again.If histology shows that tumour has and be lower than 75% neoplastic cell, it will be selected and mark is used for doing micro-dissections so.Except that tumour infringement, control tissue should be apart from cancerous tissue 10cm at least.

Use mirVana ^TMThe miRNA separating kit (Ambion, Inc., Austin, TX USA) instructs the total RNA of separation according to manufacturer.(NanoDrop Technologies, Waltham MA) measure total rna concentration to NanoDrop 1000 Spectrophotometer.Quality monitoring to RNA is then accomplished by 2100Bioanalyzer through RNA 6000Pico LabChip kit.

Embodiment 3: microarray data

In finding research, (CA USA) instructs the optional miRNA that (difference) in the specific sample is expressed to carry out qualitative analysis according to manufacturer for Agilent Technologies, Santa Clara to use Agilent miRNA microarray platform.This chip array is taken from v.10.1 DB of Sanger, comprises the probe of 723 human miRNA.The total RNA (100ng) that obtains in the lung tissue sample that 126 LCM cut, behind the Cy3 mark, and through XDR Scan (PMT100, PMT5) scanning obtains signal.The working method of mark and hybridization is all with reference to Agilent chip specification sheets.(CA USA) will be to the raw data normalization method of monochromatic (CY3) hybridization acquisition for Agilent Technologies, Santa Clara through using the Quantile method and using GeneSpring GX10 software known in the art.

Carry out independent experiment for each measurement, the MV of each data separately that definite miRNA expression level representative obtains.

Paired t-test is not used for identifying at the optimum candidate miRNA affinity tag of gland cancer lung cancer, prognosis of squamous cell lung cancer or small cell lung cancer at fisher test (F-test) afterwards.MedCalc software is used to carry out experimenter's performance curve, and (receive operating characteristic curve ROC) analyzes to confirm specificity and the susceptibility as the candidate miRNA of the biomarker of diagnostic value.95% credibility interval is used to confirm significance.

Analyze experimental data about the array of 7 main candidate miRNA organizing intermediate energy region branch gland cancer lung cancer and normal lung tissue at the freezing excision of first aspect and see table-8 for details.

Table 8: the candidate miRNA biomarker that can distinguish gland cancer lung cancer and normal lung tissue in the excision frozen tissue

See table-9 for details about organize the intermediate energy region to divide the array of 7 main candidate miRNA of prognosis of squamous cell lung cancer and normal lung tissue to analyze experimental data at the freezing excision of second aspect.

Table 9: the candidate miRNA biomarker that can distinguish lung squamous cancer and normal lung tissue in the excision frozen tissue

See table-10 for details about organize the intermediate energy region to divide the array of 7 main candidate miRNA of small cell lung cancer and normal lung tissue to analyze experimental data at the freezing excision of the third aspect.

Table 10: the candidate miRNA biomarker that can distinguish small cell lung cancer and normal lung tissue in the excision frozen tissue

See table-11 for details about organize the intermediate energy region to divide the array of 7 main candidate miRNA of prognosis of squamous cell lung cancer and gland cancer lung cancer to analyze experimental data at the freezing excision of fourth aspect.

Table 11: the candidate miRNA biomarker that can distinguish lung squamous cancer and gland cancer lung cancer in the excision frozen tissue

Organize the intermediate energy region to divide the array of 7 main candidate miRNA of small cell lung cancer and gland cancer lung cancer to analyze experimental data about the freezing excision aspect the 5th and see table-12 for details.

Table 12: the candidate miRNA biomarker that can distinguish small cell lung cancer and gland cancer lung cancer in the excision frozen tissue

Organize the intermediate energy region to divide the array of 7 main candidate miRNA of small cell lung cancer and prognosis of squamous cell lung cancer to analyze experimental data about the freezing excision aspect the 6th and see table-13 for details.

Table 13: the candidate miRNA biomarker that can distinguish small cell lung cancer and lung squamous cancer in the excision frozen tissue

Embodiment 4: the checking of microarray data in paraffin-embedded operation tissue

Be the miRNA expression data that checking obtains, instruct according to manufacturer and use the real-time quantitative RT-PCR of having set up, its adopt TaqMan microRNA assay method (Applied Biosystems, Foster City, CA, USA).To having verified hsa-miR-205 (SEQ ID NO:1) in the 215 routine paraffin-embedded excision tissues successively; Hsa-miR-25 (SEQ ID NO:2); Hsa-miR-27a (SEQ ID NO:3); Hsa-miR-29a (SEQ ID NO:4), the expression (table 1) of hsa-miR-29b (SEQ ID NO:5) hsa-miR-34a (SEQ ID NO:6) and hsa-miR-375 (SEQ ID NO:7).The detection of carrying out simultaneously that little RNA U47 is expressed is used as standardized quality monitoring.Each tests triplicate.

Briefly, instruct, adopt Taqman microRNA RT test kit to carry out reverse transcription and measure according to Applied Biosystem.Total RNA of 100ng is being comprised 1X rt damping fluid, 1X RT primer, 1nM dNTP carries out reverse transcription in the 15 microlitre RT mixed solutions of 4U RNase suppressor factor and 50U MultiScribe reversed transcriptive enzyme.(Thermal cycler alpha engine Bio-rad) goes up reaction, and steering routine is following: 16 ° of C, 30 minutes then these RT mixed solutions to be placed on the PCR appearance; 42 ° of C, 30 minutes; 85 ° of C, 5 minutes.And adopt TaqMan Universal PCR Master Mix test kit and Taqman microRNA mensuration test kit to carry out the mensuration of quantitative PCR according to the guidance of Applied Biosystem.

The RT product of 2 microlitres is at 1X TaqMan Universal PCR Master Mix, and No AmpErase UNG increases among the 1X TaqMan MicroRNA Assay mix.On Roch Light Cycling 480 appearance, carry out real-time fluorescence quantitative PCR, steering routine is following: 96 ° of C, 5 minutes initial heats; 40 or 50 circulations under 95 ° of C then, 15 seconds; 60 ° of C, 60 seconds.The Cp value is calculated and is got through the secondary derivatization method by LC480 software.The Cp value of last according to standard sample is carried out absolute quantitation to miRNA.

Paired t-test is not used to identify the differential expression of miRNA afterwards at fisher test (F-test).MedCalc software is used to carry out experimenter's performance curve, and (receive operating characteristic curve ROC) analyzes to confirm specificity and the susceptibility as the candidate miRNA of the biomarker of diagnostic value.95% credibility interval is used to confirm significance.

Experimental data about the candidate miRNA of 4 empirical tests organizing intermediate energy region branch gland cancer lung cancer and lung healthy tissues at the paraffin-embedded excision of first aspect sees table-14 for details.Wherein preferred hsa-miR-27a (SEQ ID NO:3), hsa-miR-29a (SEQ ID NO:4) and hsa-miR-34a (SEQ ID NO:6) use the runic mark.

Table 14: the checking miRNA biomarker that can distinguish lung squamous cancer and normal lung tissue in the excision paraffin organization

About organize the intermediate energy region to divide the experimental data of candidate miRNA of 4 empirical tests of prognosis of squamous cell lung cancer and lung healthy tissues to see table-15 for details at the paraffin-embedded excision of second aspect.Preferred hsa-miR-205 (SEQ ID NO:1), hsa-miR-25 (SEQ ID NO:2) and hsa-miR-29a (SEQ ID NO:3) use the runic mark.

Table 15: the checking miRNA biomarker that can distinguish small cell lung cancer and normal lung tissue in the excision paraffin organization

About organize the intermediate energy region to divide the experimental data of candidate miRNA of 5 empirical tests of small cell lung cancer and lung healthy tissues to see table-16 for details at the paraffin-embedded excision of the third aspect.Preferred hsa-miR-29a (SEQ ID NO:3) and hsa-miR-375 (SEQ ID NO:7) use the runic mark.

Table 16: the checking miRNA biomarker that can distinguish small cell lung cancer and normal lung tissue in the excision paraffin organization

About organize the intermediate energy region to divide the experimental data of candidate miRNA of 4 empirical tests of prognosis of squamous cell lung cancer and gland cancer lung cancer to see table-17 for details at the paraffin-embedded excision of fourth aspect.Preferred hsa-miR-205 (SEQ ID NO:1), hsa-miR-25 (SEQ ID NO:2) and hsa-miR-375 (SEQ ID NO:7) use the runic mark.

Table 17: the checking miRNA biomarker that can distinguish lung squamous cancer and gland cancer lung cancer in the excision paraffin organization

Organize the intermediate energy region to divide the experimental data of candidate miRNA of 5 empirical tests of small cell lung cancer and gland cancer lung cancer to see table-18 for details about the paraffin-embedded excision aspect the 5th.Preferred hsa-miR-205 (SEQ ID NO:1), hsa-miR-34a (SEQ ID NO:6) and hsa-miR-375 (SEQ ID NO:7) use the runic mark.

Table 18: the checking miRNA biomarker that can distinguish small cell lung cancer and gland cancer lung cancer in the excision paraffin organization

Organize the intermediate energy region to divide the experimental data of candidate miRNA of 5 empirical tests of small cell lung cancer and prognosis of squamous cell lung cancer to see table-19 for details about the paraffin-embedded excision aspect the 6th.Preferred hsa-miR-205 (SEQ ID NO:1), hsa-miR-34a (SEQ ID NO:6) and hsa-miR-375 (SEQ ID NO:7) use the runic mark.

Table 19: the checking miRNA biomarker that can distinguish small cell lung cancer and lung squamous cancer in the excision paraffin organization

The result who is obtained confirms the high specific adjusting that miRNA expresses in lung cancer.Therefore; The corresponding inferior collection of miRNA as herein described is represented unique miRNA expression characteristic; The expression pattern analysis that is used for lung cancer, it not only makes can differentiate oncogenesis state (cancerogenous state) itself, and feasiblely can distinguish dissimilar lung tumors.

Embodiment 5: the method that quantizes the miRNA biomarker

Through use the TaqMan microRNA measure (Agilent Technologies, Santa Clara, CA, real-time quantitative RT-PCR USA) instructs the optional miRNA affinity tag that (difference) in the specific sample is expressed to carry out quantitative analysis according to manufacturer.

Particularly preferably, said miRNA biomarker quantitatively can realize (Fig. 1) through in situ hybridization.Said method is following:

(b) on slide glass, prepare tissue slice;

(d) at microscopically or by digital pathology answer method the expression of miRNA is carried out quantitatively;

(e) in described one or more target cells, confirm multiple nucleic acid molecule expression level, every kind of nucleic acid molecule miRNA sequence of all encoding;

(g) through contrasting in step (e) and the expression level separately that obtains (f); From said multiple nucleic acid molecule, identify one or more nucleic acid molecule of differential expression in target cell and control cells; The nucleic acid molecule of wherein said one or more differential expressions is represented the expression of nucleic acid characteristic like this paper definition together, and it is the indication that has gland cancer lung cancer, prognosis of squamous cell lung cancer or small cell lung cancer.

For the expression level that the said labeled nucleic acid molecule thing of standard code miRNA sequence obtains, the expression of nucleic acid molecule of said coding hsa-miR-24 (SEQ ID NO:8) maybe be preferred, and it is stably express in the knot rectal tissue.And the negative control of the expression level that obtains for the said labeled nucleic acid molecule thing of coding miRNA sequence, the expression of nucleic acid molecule of described coding hsa-122 (SEQ ID NO:9) maybe be preferred, and it is not expressed in the knot rectal tissue.

The probe that is used to hybridize is that hybridization is in the comprehensive infinite sequence (GGGGGTCCTATATGGCTCCACTTCTCCCCC) of the quilt of the target miRNA that comprises 30 residues.Said residue sequence is used the runic mark.Probe is marked on the fluorescent group at 5 ' end.Single or a plurality of probes can parallelly be hybridized with independent optical dye.The probe sequence that is used in situ hybridization among the present invention provides at table-20.

Described residue 5 ' hairpin structure (GGGGG-CCCCC to) is in order to be stabilized in the hybridization between probe and the target miRNA and to improve the hybridization specificity.In the hybridization, same principle can be used for designing the probe of other miRNA affinity tags in position.

Table 20: in situ hybridization probe

The present invention who describes for example in this article can suitably carry out under the condition that does not have the special any element that discloses of this paper, restriction.Therefore, for example term " comprises ", " comprising " " contain " etc. and should have broad sense and unrestricted.In addition; Term and expression that this paper uses are used to describe the present invention and unrestricted meaning; And do not use these terms and meaning that express to get rid of any characteristic shown in it and description or its a part of Equivalent, still should recognize in the scope of the invention of asking for protection and to carry out various modifications.Therefore, although should understand the special announcement of the present invention being carried out through embodiment and optional characteristic, those skilled in the art can make amendment and change the present invention, and this modification and change are thought within the scope of the invention.

This paper extensively reaches and has briefly described the present invention.A part of the present invention has also been formed in each the narrower subordinate concept and the inferior upper set that fall in the upper description scope.This comprises the negative restriction of from upper, removing any theme with conditioned disjunction to upper description of the present invention, and whether the theme of no matter being removed is quoted from this article especially.

Other embodiment is in following claim scope.In addition, when characteristic of the present invention or all respects were described with Ma Kushi prescription formula, those skilled in the art can recognize that the present invention also is described with any each member or the inferior prescription formula of member of Ma Kushi group.

Claims

1. be used to identify the diagnostic kit of the molecule marker of one or more Mammals target cells that show lung cancer, said test kit comprises multiple nucleic acid molecule, every kind of nucleic acid molecule microrna sequences of all encoding,

One or more of wherein said multiple nucleic acid molecule are differential expression in said target cell and in one or more control cells; And the nucleic acid molecule of wherein said one or more differential expressions is represented the expression of nucleic acid characteristic together; Said characteristic is a lung cancer; And/or the indication of different subtype lung cancer existence

Wherein different lung cancer is made up of gland cancer lung cancer, prognosis of squamous cell lung cancer or small cell lung cancer.

2. the test kit of claim 1, wherein said lung cancer is gland cancer lung cancer.

3. claim 1 or 2 test kit, wherein said expression of nucleic acid biomarker can comprise at least four kinds of nucleic acid molecule.

4. the test kit of claim 1-3, wherein said expression of nucleic acid biomarker comprises the nucleic acid molecule of at least a coding microrna sequences, and it is expressed in one or more target cells with in one or more normal control cells, comparing and is raised; And comprising the nucleic acid molecule of at least a coding microrna sequences, it is expressed in one or more target cells with in one or more normal control cells, comparing and is reduced.

5. each test kit of claim 1-4, wherein said expression of nucleic acid biomarker comprise any one or the multiple nucleic acid molecule of coding hsa-miR-27a, hsa-miR-29a, hsa-miR-34a and hsa-miR-375.

6. the test kit of claim 5; Wherein in said one or more target cells with in said one or more normal control cells, compare; The expression of hsa-miR-34a and hsa-miR-375 is raised, and the expression of any one or the multiple nucleic acid molecule of coding hsa-miR-27a and hsa-miR-29a is reduced.

7. the test kit of claim 5, wherein said expression of nucleic acid biomarker comprise any one or the combination of multiple nucleic acid of coding hsa-miR-34a/hsa-miR-27a and hsa-miR-34a/hsa-miR-29a.Particularly, said expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-29a, hsa-miR-27a and hsa-miR-34a.

8. the test kit of claim 1, wherein said lung cancer is prognosis of squamous cell lung cancer.

9. claim 1 or 8 test kit, wherein said expression of nucleic acid biomarker can comprise at least four kinds of nucleic acid molecule.

10. the test kit of claim 1 or 8-9, wherein said expression of nucleic acid biomarker comprises the nucleic acid molecule of at least a coding microrna sequences, and it is expressed in one or more target cells with in one or more normal control cells, comparing and is raised; And comprising the nucleic acid molecule of at least a coding microrna sequences, it is expressed in one or more target cells with in one or more normal control cells, comparing and is reduced.

11. each test kit of claim 1 or 8-10, wherein said expression of nucleic acid biomarker comprise any one or the multiple nucleic acid molecule of coding sa-miR-205, hsa-miR-25, hsa-miR-29a and hsa-miR-375.

12. the test kit of claim 11; Wherein in said one or more target cells with in said one or more normal control cells, compare; The expression of hsa-miR-205 and hsa-miR-25 is raised, and the expression of any one or the multiple nucleic acid molecule of coding hsa-miR-29a and hsa-miR-375 is reduced.

13. the test kit of claim 11, wherein said expression of nucleic acid biomarker comprise any one or the combination of multiple nucleic acid of coding hsa-miR-205/hsa-miR-29a, hsa-miR-25/hsa-miR-29a, hsa-miR-205/hsa-miR-375, hsa-miR-25/hsa-miR-375.Particularly, said expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-205, hsa-miR-25 and hsa-miR-29a.

14. the test kit of right 1, wherein said lung cancer is small cell lung cancer.

15. the test kit of claim 1 or 14, wherein said expression of nucleic acid biomarker can comprise at least five kinds of nucleic acid molecule.

16. the test kit of claim 1 or 14-15, wherein said expression of nucleic acid biomarker comprises the nucleic acid molecule of at least a coding microrna sequences, and it is expressed in one or more target cells with in one or more normal control cells, comparing and is raised; And comprising the nucleic acid molecule of at least a coding microrna sequences, it is expressed in one or more target cells with in one or more normal control cells, comparing and is reduced.

17. each test kit of claim 1 or 14-16, wherein said expression of nucleic acid biomarker comprise any one or the multiple nucleic acid molecule of coding hsa-miR-25, hsa-miR-27a, hsa-miR-29a, hsa-miR-29b and hsa-miR-375.

18. the test kit of claim 17; Wherein in said one or more target cells with in said one or more normal control cells, compare; The expression of hsa-miR-25 and hsa-miR-375 is raised, and the expression of any one or the multiple nucleic acid molecule of coding hsa-miR-27a, hsa-miR-29a and hsa-miR-29b is reduced.

19. the test kit of claim 17, wherein said expression of nucleic acid biomarker comprise any one or the combination of multiple nucleic acid of coding hsa-miR-25/hsa-miR-27a, hsa-miR-375/hsa-miR-27a, hsa-miR-25/hsa-miR-29a and hsa-miR-375/hsa-miR-29a.Particularly, said expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-29a and hsa-miR-375.

20. the test kit of claim 1-19 is further used for prognosis of squamous cell lung cancer and gland cancer lung cancer are differentiated.

21. the test kit of claim 1 or 20, wherein said expression of nucleic acid biomarker can comprise at least four kinds of nucleic acid molecule.

22. the test kit of claim 1 or 20-21, wherein said expression of nucleic acid biomarker comprises the nucleic acid molecule of at least a coding microrna sequences, and it is expressed in one or more target cells with in one or more control cells, comparing and is raised; And comprising the nucleic acid molecule of at least a coding microrna sequences, it is expressed in one or more target cells with in one or more control cells, comparing and is reduced.

23. each test kit of claim 1 or 20-22, wherein said expression of nucleic acid biomarker comprise any one or the multiple nucleic acid molecule of coding hsa-miR-205, hsa-miR-25, hsa-miR-27a and hsa-miR-375.

24. the test kit of claim 23; Wherein in said one or more target cells with in said one or more control cells, compare; The expression of hsa-miR-205, hsa-miR-25 and hsa-miR-27a is raised, and the expression of a kind of nucleic acid molecule of coding hsa-miR-375 is reduced.

25. comprising any one or the multiple nucleic acid of coding hsa-miR-205/hsa-miR-375 and hsa-miR-25/hsa-miR-375, the test kit of claim 23, wherein said expression of nucleic acid biomarker make up.Particularly, said expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-205, hsa-miR-25 and hsa-miR-375.

26. the test kit of claim 1-19 is further used for small cell lung cancer and gland cancer lung cancer are differentiated.

27. the test kit of claim 1 or 26, wherein said expression of nucleic acid biomarker can comprise at least six kinds of nucleic acid molecule.

28. the test kit of claim 1 or 26-27, wherein said expression of nucleic acid biomarker comprises the nucleic acid molecule of at least a coding microrna sequences, and it is expressed in one or more target cells with in one or more control cells, comparing and is raised; And comprising the nucleic acid molecule of at least a coding microrna sequences, it is expressed in one or more target cells with in one or more control cells, comparing and is reduced.

29. each test kit of claim 1 or 26-28, wherein said expression of nucleic acid biomarker comprise any one or the multiple nucleic acid molecule of coding hsa-miR-25, hsa-miR-27a, hsa-miR-29a, hsa-miR-29b, hsa-miR-34a and hsa-miR-375.

30. the test kit of claim 29; Wherein in said one or more target cells with in said one or more control cells, compare; The expression of hsa-miR-25 and hsa-miR-375 is raised, and the expression of any one or the multiple nucleic acid molecule of coding hsa-miR-27a, hsa-miR-29a, hsa-miR-29b and hsa-miR-34a is reduced.

31. the test kit of claim 1 or 26-30, wherein said expression of nucleic acid biomarker comprise any one or the combination of multiple nucleic acid of coding hsa-miR-25/hsa-miR-27a, hsa-miR-375/hsa-miR-27a, hsa-miR-25/hsa-miR-29a and hsa-miR-375/hsa-miR-29a.Particularly, said expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-25, hsa-miR-34a and hsa-miR-375.

32. the test kit of claim 1-19 is further used for small cell lung cancer and prognosis of squamous cell lung cancer are differentiated.

33. the test kit of claim 32, wherein said expression of nucleic acid biomarker can comprise at least six kinds of nucleic acid molecule.

34. the test kit of claim 1 or 32-33, wherein said expression of nucleic acid biomarker comprises the nucleic acid molecule of at least a coding microrna sequences, and it is expressed in one or more target cells with in one or more control cells, comparing and is raised; And comprising the nucleic acid molecule of at least a coding microrna sequences, it is expressed in one or more target cells with in one or more control cells, comparing and is reduced.

35. each test kit of claim 1 or 32-34, wherein said expression of nucleic acid biomarker comprise any one or the multiple nucleic acid molecule of coding hsa-miR-205, hsa-miR-27a, hsa-miR-29a, hsa-miR-29b, hsa-miR-34a and hsa-miR-375.

36. the test kit of claim 35; Wherein in said one or more target cells with in one or more control cells, compare; The expression of hsa-miR-375 is raised, and the expression of any one or the multiple nucleic acid molecule of coding hsa-miR-205, hsa-miR-27a, hsa-miR-29a, hsa-miR-29b and hsa-miR-34a is reduced.

37. comprising any one or the multiple nucleic acid of coding hsa-miR-375/hsa-miR-27a and hsa-miR-375/hsa-miR-29a, the test kit of claim 35, wherein said expression of nucleic acid biomarker make up.Particularly, said expression of nucleic acid biomarker comprises a kind of nucleic acid combination of coding hsa-miR-29b and hsa-miR-375.

38. be used to identify the method for one or more Mammals target cells that show gland cancer lung cancer, prognosis of squamous cell lung cancer or small cell lung cancer, said method comprises:

(b) on slide glass, prepare tissue slice;

39. the method for claim 38 is further used for distinguishing the lung cancer that is selected from gland cancer lung cancer, prognosis of squamous cell lung cancer and small cell lung cancer.

40. the method for prevention or treatment lung cancer in one or more Mammals target cells, said method comprises:

(a) in one or more target cells, identify the expression of nucleic acid characteristic through the method for using claim 53-54; And

The expression of one or more nucleic acid molecule of the coding microrna sequences that (b) the said expression of nucleic acid characteristic of change is comprised in said one or more cells; Said change is carried out in such a way; Be that its expression that is expressed in the nucleic acid molecule that is raised in said one or more target cells is reduced, and its expression that is expressed in the nucleic acid molecule of being reduced in said one or more target cells is raised.

41. be used for preventing and/or treating the lung cancer drugs compsn one or more Mammals target cells, said compsn comprises one or more nucleic acid molecule, every kind of equal encoding sequence of nucleic acid molecule,

Said sequence with as 1-55 each defined it is expressed the coded microrna sequences of nucleic acid molecule raised part is complementary at least in said one or more target cells, and/or said sequence corresponding to as claim 1-40 each defined it expresses coded microrna sequences of nucleic acid molecule of being reduced in said one or more target cells.

42. the pharmaceutical composition of claim 41 prevents and/or treats the purposes in the lung cancer drugs in preparation.