CN102933719B

CN102933719B - Compositions and methods for microrna expession profiling in plasma of colorectal cancer

Info

Publication number: CN102933719B
Application number: CN201080064801.4A
Authority: CN
Inventors: 李兆勇; 吴莹; 朱虹光; 李健; 任一萍
Original assignee: Fudan University
Current assignee: SHANGHAI LABWAY CLINICAL LABORATORY Co.,Ltd.
Priority date: 2009-12-24
Filing date: 2010-12-24
Publication date: 2015-05-06
Anticipated expiration: 2030-12-24
Also published as: CN102933719A

Abstract

The present invention relates to compositions and methods for microRNA (miRNA) expression profiling in plasma of colorectal cancer. In particular, the invention relates to a diagnostic kit of molecular markers in blood for diagnosing colorectal cancer, monitoring the cancer therapy and/or treating colorectal cancer, the kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in plasma of colorectal cancer and healthy control plasma, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of colorectal cancer. The invention further relates to corresponding methods using such nucleic acid expression signatures for identifying colorectal cancer as well as for preventing or treating such a condition. Finally, the invention is directed to a pharmaceutical composition for the prevention and/or treatment of colorectal cancer.

Description

For composition and the method for the micro-RNA expression spectrum analysis in plasma of colorectal cancer

Invention field

The present invention relates to the composition for microRNA (microRNA) expression pattern analysis in colorectal cancer (colorectal cancer) blood plasma and method.

Background of invention

Colorectal cancer (CRC) was most important human cancer, and just there were ~ 1041200 new cases in the whole world in 2008.It is the third-largest most common cancer and the fourth-largest reason causing cancer mortality in the world.(Gry.R.et al.(1997))Curr Probl Cancer 21,233-300；Petersen,G.M.et al.(1999)Cancer 86,254002550)。If can phases diagnostic in early days, CRC can cure.In the stage in early days, most patients is the clinical symptom not having disease, is therefore necessary the examination carrying out early stage CRC.Evidence shows that the discovery of early stage colorectal cancer can reduce incidence and the mortality ratio (Anwar, R. (2006) Digestive and Liver Disease 34,279-282) of disease to a great extent.

The feature of initial CRC is the hyperplasia (atypia) of colonic epithelium, and it is first transformed into struvite adenomatous polyp, is then transformed into the aberrant nascent thing adenoma (i.e. innocent tumour) of Colon and rectum mucous membrane.Under normal circumstances, the adenoma (incidence of 60 years old patient is 60-70%) only having small portion to be formed develops into adenocarcinoma.CRC case more than 95% shows as gland cancer (Muto, T.et al. (1975) Cancer36,2251-2270; Fearon, E.R.and Vogelstein, B. (1990) Cell 61,759-767).

Existing CRC standard sieve checking method comprises Sigmoidoscope and fecal occult blood test.But these two tests have serious defect.Colonoscopy is effective, but majority are unwilling to accept this to be checked because of its high cost, the very large discomfort brought and potential more side effects.On the other hand, fecal occult blood is tested simple and is spent low but relatively not accurate enough.But, also do not make it possible to the specific molecule marker reliably diagnosing CRC at present, especially for the malignant tumour that the CRC and/or benign adenoma that show as gland cancer develop into.

Therefore, the discovery of new biomarker has most important clinical efficacy, especially the development of these mark early diagnosis tumours, to make cancer obtain early treatment and to avoid unnecessary surgical intervention.Ideally, these marks should make it possible to find to be in the cancer that in-situ techniques or biopsy or the microscopical analysis of excision thing can not find malignant cell period.

Some diagnostic methods are also because it analyzes the reliability and/or accuracy that to affect detection based on single molecule marker and limited.In addition, single mark can not detailed forecasts to be correlated with stage in latent period, tumour progression and analogue usually.Therefore, the discovery of alternative molecule marker and the detection mode of needing is to overcome these obstacles.

A ways of addressing this issue may based on little modulability RNA molecule especially microRNA (miRNA), and the length it constituting the small-sized untranslated of the endogenous expression of evolution conservative is the RNA of 20-25 Nucleotide (nt).Since finding before 10 years, think that it can mediate the expression of target mRNA and therefore have the critical function participating in cell generation, differentiation, proliferation and apoptosis.(Bartel,D.P.(2004)Cell 116,281-297,Ambros,V.(2004)Nature 431,350-355；He.L.etal.(2004)Nat Rev Genet 5,522-531)。In addition, as 25 kinds of cancer biomarkers, miRNA is due to very stable in vitro and long-lived in vivo and have more advantage (Lu, J.etal. (2005) Nature 435,834-838 compared to mRNA; Lim, L.P.et al. (2005) Nature 433,769-773).

MiRNA derives from the loop-stem structure precursor (pre-miRNA) be processed into by RNase III Drosha in primary transcription process.After being transported to endochylema, another kind is called that pre-miRNA hairpin loop is cut into short double-strand RAN (dsRNA) by the RNaseIII of Dicer, and wherein chain becomes the ripe miRNA in miRNA albumen (miRNP).MiRNA guides miRNP can play target mRNA (Bartel, D.P. (2004) Cell 23,281-292 of function to it; He, L.and Hannon, G.J. (2004) Nat Rev Genet 5,522-531).

According to the complementarity of miRNA and target, the bootable different regulation process of miRNA.The target mRNA highly complementary with miRNA is cut by the structural specificity identical with RNA interfering (RNAi) ground.Therefore, in this case, miRNA play function as siRNA (siRNA).The target mRNA lower with miRNA complementarity is same to be directed into cell degradation path or to be translated suppression when not affecting mRNA level in-site.But miRNA suppresses the mechanism of the translation of target mRNA still to have dispute.Available data shows that miRNA can be used as oncogene or tumor suppressor gene and plays a role in cancer, and the mir-17-92 of such as, in cancer overexpression may play function as oncogene and by lowering tumor suppressor gene and/or controlling the gene of cytodifferentiation or apoptosis thus promote the development of cancer.Equally, the low expression of let-7a, it plays tumor suppressor gene function, can suppress cancer (Zhang, B. (2007) Dev Biol 302,1-12) by modulate tumor gene and/or the gene controlling cytodifferentiation or apoptosis.Show that they occur in cancer and play a role in development.

High-throughput miRNA quantitative technique such as miRNA microarray, the overall miRNA measured etc. as the whole oncogene group of research of the TaqMan miRNA based on real-time RT-PCR composes and provides powerful measure.It is abnormal that increasing evidence shows that multiple miRNA comprises performance in leukemia, lymphoma, glioblastoma multiforme, colorectal carcinoma, lung cancer, mammary cancer, prostate cancer, thyroid carcinoma, liver cancer and ovarian cancer at human cancer, and expression is different in healthy tissues with cancerous tissue.(Zhang,L.25(2008)Adv Exp Med Biol 622,69-78)。Therefore, miRNA spectrum is used as the mark finding kinds cancer, shows that it will contribute to determining molecular diagnosis, prognosis and treatment further.MiRNA abnormal in human cancer shows the potentiality of these miRNA as biomarker and molecular therapy target.

Multiple may in the sample of type, blood is considered to the ideal sample of examination excessive risk individuality, because it can be collected by the minimum mode of infection, can early discovery, diagnosis, monitoring and effective Therapeutic cancer.Show that miRNA that tumour originates is by being present in human plasma or serum with highly stable form under the effect protection of endogenous RNase.The miRNA in these blood plasma or serum tumor source can be used as the biomarker of discovery cancer and reaches the level that can survey.In addition, the miRNA horizontal correlation of blood plasma and serum is large, show that blood plasma or serum sample can be used as the samples selection type (Mitchell of the clinical biomarker using miRNA as cancer prognosis, P.S.et al. (2008) Proc Natl Acad Sci USA 105,10513-10518; Gilad, S.et al. (2008) PLoSONE 3, e3148; Chen, X.et al. (2008) Cell Res 18,997-101006).

Some study miRNA express spectra (Chen, X. (2008) the Cell Res 18,997-1006 in the blood plasma or serum having reported Human Colorectal Cancer case; Ng, E.K.O. (2009) Gut 58,1375-1381; Huang, Z.2009, Int J Cancer, published on line).Find more than 100 kinds of circulation miRNA (Mitchell in healthy individuals, P.S. (2008) Proc Natl Acad Sci USA105,10513-10518), and this spectrum composes a great difference with the miRNA of the Patients with Colorectal Cancer with kinds of tumors specificity miRNA.The people such as Chen demonstrate the 69 kinds of miRNA (Chen, X. (2008) Cell Res18,997-1006) being present in and not being present in Normal group serum in Patients with Colorectal Cancer serum.In addition, they have found to be present in colorectal cancer and the unique express spectra not being present in 14 kinds of miRNA in other cancer group (lung cancer) serum.The research of the people such as Ng shows that miR-92 significantly raises in the blood plasma of CRC case, and has the potentiality (E.K.O. (2009) Gut 58,1375-1381) of the Noninvasive molecule marker as CRC examination.In addition, the people such as Huang find that blood plasma miR-29a has the very large potentiality (Huang, Z.2009, Int J Cancer.published on line) of the new Noninvasive biomarker as early discovery CRC.But, another research finds that has-miR-92a significantly declines in Patients with Acute Leukemia blood plasma, and miR-92a/miR-638 ratio has the very large potentiality (Tanaka as detecting leukemic true tumor mark in blood plasma, M.et al. (2009) PLoS ONE 4, e5532).But owing to lacking enough Sensitivity and Specificity, single miRNA is not suitable as and diagnoses biomarker for clinical application accurately.

Therefore, be still necessary in discovery one group of Patients with Colorectal Cancer blood plasma or in serum, diagnose miRNA to mark.The determination that the miRNA based on blood in conjunction with multiple miRNA biomarker composes (feature) can make Noninvasive, fast, accurately and the method for the determination Patients with Colorectal Cancer of economy becomes possibility.In addition, also need in excessive risk individuality for the early detection of the correlation method of commitment and colorectal cancer examination, cancer return and/or monitoring cancer therapy always.

Purpose of the invention and overview

The object of this invention is to provide for Colorectal Cancer Diagnosis, the treatment of monitoring cancer, and/or the novel method for the treatment of colorectal cancer, wherein by measuring the multiple nucleic acids molecule in blood, often kind of nucleic acid molecule is all encoded microRNA (miRNA) sequence, one or more of wherein said multiple nucleic acids molecule in the blood plasma of colorectal cancer by analysis compared with normal healthy controls and/or and healthy individuals, hepatocellular carcinoma compares differential expression with lung cancer, the nucleic acid molecule of one or more differential expression wherein said represents expression of nucleic acid feature together, this expression of nucleic acid feature is the indication that colorectal cancer exists, wherein said expression of nucleic acid feature comprises tumour correlated characteristic (tumor-related signature) and plasma specific feature (plasma-specific signature).

In addition, the object of this invention is to provide the correlation method for differentiating one or more expression of nucleic acid feature in the blood representing colorectal cancer.More specifically, the object of this invention is to provide and distinguish the method for colorectal cancer for comparing with normal healthy controls and/or healthy individuals, hepatocellular carcinoma and lung cancer.

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

In first aspect, the present invention relates to for differentiating that one or more shows the diagnostic kit of the blood Middle molecule marker of the target plasma of colorectal cancer, described test kit comprises multiple nucleic acids molecule, often kind of nucleic acid molecule encoding microrna sequences, one or more differential expression in target plasma and in one or more contrast blood plasma of wherein said multiple nucleic acids molecule, the nucleic acid molecule of one or more differential expression wherein said comes from tumour and is correlated with or plasma specific feature, the nucleic acid molecule of one or more differential expression wherein said represents expression of nucleic acid feature together, described expression of nucleic acid feature is the indication that colorectal cancer exists.

The expression of nucleic acid feature limited herein can comprise at least 35 kinds of nucleic acid molecule, preferably at least 12 kinds of nucleic acid molecule, particularly preferably at least 6 kinds of nucleic acid molecule.

In preferred embodiments, described expression of nucleic acid feature comprises the nucleic acid molecule of at least one coding microrna sequences, and its expression is raised in one or more target plasma compared with one or more normal healthy controls; And comprising the nucleic acid molecule of at least one coding microrna sequences, its expression is lowered in one or more target plasma compared with one or more normal healthy controls.

In preferred embodiments, described expression of nucleic acid feature comprises codes for tumor correlated characteristic hsa-miR-409-3p, hsa-miR-25, hsa-miR-93, hsa-miR-96, hsamiR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsa-miR-486-5p, hsamiR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa-miR-107, hsa-miR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185; And plasma specific feature hsa-mir-671-3p, hsa-mir-16-2*, hsa-miR-30c-1*, hsa-miR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsamiR-363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa-miR-130a, hsa-miR-331-3p, hsa-miR-345; With any one or the multiple nucleic acids molecule of internal stability contrast hsa-miR-1238 and hsa-miR-1228.

Particularly preferably, compared with one or more normal healthy controls, in one or more target plasma described: coding hsa-miR-409-3p and hsa-mir-671-3p the expression of any one or multiple nucleic acids molecule raised, and the hsa-miR-25 that encodes, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsa-miR-486-5p, hsa-miR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa-miR-107, hsamiR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185 ' hsa-mir-16-2*, hsa-miR-30c-1*, hsa-miR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsa-miR-363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa-miR-130a, any one or the multiple nucleic acids developed by molecule of has-miR-331-3p and hsa-miR-345 are lowered, hsa-miR-1238 and hsa-miR-1228 does not change.

In a more preferred embodiment, expression of nucleic acid feature comprises any one or the multiple nucleic acids molecule of codes for tumor correlated characteristic hsamiR-409-3p, hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451 and coding plasma specific feature hsa-mir-671-3p, hsa-mir-16-2*, hsa-miR-30c-1*, hsa-miR-548c-5p.

Particularly preferably, compared with one or more normal healthy controls, in one or more target plasma: one or more nucleic acid molecule any of coding hsa-miR-409-3p and hsa-mir-671-3p is expressed and raised; And any one or the multiple nucleic acids developed by molecule of encode hsa-miR-25, hsamiR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsamir-16-2*, hsa-miR-30c-1* and hsa-miR-548c-5p are lowered.

In another preferred embodiment, expression of nucleic acid feature comprises any one or the multiple nucleic acids molecule of codes for tumor correlated characteristic hsa-miR-409-3p, hsa-miR-25, hsa-miR-93, hsa-miR-96 and plasma specific feature hsa-mir-671-3p, hsa-mir-16-2*.

Particularly preferably, compared with one or more normal healthy controls, in one or more target plasma: the expression of any one or the multiple nucleic acids molecule of coding hsa-miR-409-3p and hsa-mir-671-3p is raised, and the expression of any one or the multiple nucleic acids molecule of hsa-miR-25, hsamiR-93, hsa-miR-96 and and hsa-mir-16-2* that encodes is lowered.

In particularly preferred embodiments, expression of nucleic acid feature comprises coding hsa-miR-409-3p/hsa-miR-16-2*, hsa-miR-409-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-548c-5p, hsa-miR-671-3p/hsa-miR-16-2*, hsa-miR-671-3p/hsa-miR-30c-1*, hsa-miR-671-3p/hsa-miR-342-3p, hsa-miR-671-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-301a, hsa-miR-671-3p/hsa-miR-345, hsa-miR-409-3p/hsa-miR-345, hsa-miR-409/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-25, any one or the multiple nucleic acids molecular combinations of hsa-miR-409-3p/hsa-miR-93 and hsa-miR-671-3p/hsa-miR-93.

In second aspect, the present invention relates to for the diagnostic kit differentiated by colorectal cancer and healthy individuals, hepatocellular carcinoma and lung cancer.Described test kit comprises multiple nucleic acids molecule, often kind of nucleic acid molecule encoding microrna sequences, one or more differential expression in target plasma and in one or more healthy individuals, hepatocellular carcinoma and lung cancer of wherein said multiple nucleic acids molecule, and the nucleic acid molecule of one or more differential expression wherein said represents expression of nucleic acid feature together, described expression of nucleic acid feature is the indication that colorectal cancer exists.

The expression of nucleic acid feature limited herein can comprise at least 23 kinds of nucleic acid molecule, preferably at least 18 kinds of nucleic acid molecule, and more preferably at least 6 kinds of nucleic acid molecule.

In preferred embodiments, described expression of nucleic acid feature comprises the nucleic acid molecule of at least one coding microrna sequences, and it is expressed to compare with one or more healthy individuals, hepatocellular carcinoma and lung cancer in one or more target plasma and is raised; And comprising the nucleic acid molecule of at least one coding microrna sequences, it is expressed to compare with one or more healthy individuals, hepatocellular carcinoma and lung cancer in one or more target plasma and is lowered.

In an even more preferred embodiment, expression of nucleic acid feature comprises coding: tumour correlated characteristic hsa25miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-7, hsa-miR-196b, hsa-miR-93, hsa-miR-486-5p, hsa-miR-25, hsa-miR-92a, hsa-miR-19b; Any one or the multiple nucleic acids molecule of plasma specific feature hsa-miR-671-3p and hsa-miR-16-2*, hsa-miR-92b, hsa-miR-129*, hsa-miR-563, hsamiR-602, hsa-miR-1227, hsa-miR-196a and internal stability contrast: hsa-miR-1238 and hsa-miR-1228.

Particularly preferably, with healthy individuals, hepatocellular carcinoma is compared with lung cancer, encode in one or more target plasma hsa-miR-409-3p, hsa-mir-671-3p, hsa-miR-33b*, hsa-miR-92b, hsa-miR-149, hsa-miR-129*, hsa-miR-563, hsa-miR-129-3p, hsa-miR-634, hsa-let-7b*, any one or the multiple nucleic acids developed by molecule of hsa-miR-602 and hsa-miR-1227 are raised, and the hsa-miR-16-2* that encodes, hsa-miR-7, hsa-miR-196a, hsamiR-196b, hsa-miR-486-5p, hsa-miR-93, hsa-miR-25, any one or the multiple nucleic acids developed by molecule of hsa-miR-92a and hsa-miR-19b are lowered, hsa-miR-1238 and hsa-miR-1228 does not change.

In a more preferred embodiment, expression of nucleic acid feature comprises any one or the multiple nucleic acids molecule of codes for tumor correlated characteristic has-10miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-7 and plasma specific feature hsa-miR-671-3p and hsa-miR-16-2*.

Particularly preferably, compare with lung cancer with healthy individuals, colorectal cancer, any one or the multiple nucleic acids developed by molecule of encode in one or more target plasma hsa-miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-671-3p are raised; And any one or the multiple nucleic acids developed by molecule of hsa-miR-16-2* and hsa-miR-7 that encode are lowered.

In particularly preferred scheme, expression of nucleic acid feature comprises coding hsa-miR-33b*/hsa20-miR-196a, hsa-miR-92b/hsa-miR-196a, hsa-miR-563/hsa-miR-196a, hsa-miR-1227/hsa-miR-196a, hsa-miR-129-3p/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-16-2*, hsa-miR-92b/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-196a, hsa-miR-1227/hsa-miR-16-2*, hsa-miR-129-3p/hsa-miR-196a, hsa-miR-602/hsa-miR-196a, hsa-miR-33b*/hsamiR-16-2*, hsa-miR-563/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-7, hsa-miR-33b*/hsa25-miR-7, hsa-miR-563/hsa-miR-7, hsa-miR-602/hsa-miR-16-2*, hsa-miR-129-3p/hsamiR-7, hsa-miR-92b/hsa-miR-7, hsa-miR-1227/hsa-miR-7, hsa-miR-33b*/hsa-miR-196b, hsa-miR-1227/hsa-miR-196b, any one or the multiple nucleic acids combination of hsa-miR-92b/hsa-miR-196b and hsa-miR-602/has-miR-7.

The third aspect, the present invention relates to for differentiating that one or more shows the method for the blood plasma of colorectal cancer, described method comprises: (a) determines the expression level of multiple nucleic acids molecule in one or more target plasma described, often kind of nucleic acid molecule encoding microrna sequences; B () determines the expression level of described multiple nucleic acids molecule in one or more normal healthy controls blood plasma; C respective expression level that () is obtained in step (a) and (b) by contrast, one or more nucleic acid molecule of differential expression in described target plasma and contrast blood plasma is identified from described multiple nucleic acids molecule, one or more nucleic acid molecule of wherein said differential expression represents feature (signature) defined herein together, and it is the indication that colorectal cancer exists.

In a preferred embodiment of the invention, described method comprises expression level of (a) definite kernel acid molecule combination in one or more target plasma, and often kind of nucleic acid molecule is all encoded microrna sequences, and uses specific formulae discovery; B () determines the expression level that described nucleic acid molecule combines in one or more normal healthy controls blood plasma, and use specific formulae discovery; And (c) by compare the respective expression level obtained in (a) and (b) step differentiate described in be combined in difference in one or more target plasma described, wherein all representative features of combination of one or more differential expression, it shows the existence of colorectal cancer.

In a more preferred embodiment, present method is further used for colorectal cancer and healthy individuals, hepatocellular carcinoma and lung cancer to differentiate.

Fourth aspect, the present invention relates to the method for monitoring treatment of colorectal cancer, described method comprises: (a) differentiates expression of nucleic acid feature by using method defined herein in one or more target plasma; (b) expression of one or more nucleic acid molecule of the coding microrna sequences that described expression of nucleic acid feature comprises is monitored in blood, described monitoring is carried out in such a way, namely the expression in its blood plasma is lowered after the treatment by the expression of the nucleic acid molecule raised before the treatment, and the expression in its blood plasma is raised after the treatment by the expression of the nucleic acid molecule lowered before the treatment.

5th aspect, the present invention relates to the method for preventing or treat colorectal cancer, described method comprises: (a) in blood plasma differentiates expression of nucleic acid feature by the method limited herein; (b) expression of one or more nucleic acid molecule of the coding microrna sequences that described expression of nucleic acid feature comprises is changed in blood, described change is carried out in such a way, namely its expression is lowered by the expression of the nucleic acid molecule raised in blood, and its expression is raised by the expression of the nucleic acid molecule lowered in blood.

6th aspect, the present invention relates to the pharmaceutical composition for preventing and/or treating the colorectal cancer in blood, described composition comprises one or more nucleic acid molecule, often kind of equal encoding sequence of nucleic acid molecule, described sequence is complementary at least partly with the microrna sequences that as herein defined it is expressed coded by the nucleic acid molecule that raises in from the blood plasma of colorectal cancer patients, and/or described sequence corresponds to the microrna sequences of its expression coded by the nucleic acid molecule lowered in from the blood plasma of colorectal cancer patients as herein defined.

Finally, the 7th aspect, the present invention relates to described pharmaceutical composition for the preparation of the purposes prevented and/or treated in the medicine of hepatocellular carcinoma.

Other embodiment of the present invention will become clear from the following detailed description.

Accompanying drawing is sketched

Fig. 1: schema is shown, it systematically illustrates with reference to method of the present invention to determine the basic method steps of expression characteristic, for differentiating that one or more shows the target plasma of colorectal cancer.

Fig. 2: describe according to one or more target plasma in determination colorectal cancer of the present invention, to be included in first aspect the mankind miRNA in particularly preferably expression characteristic.Also illustrate that colorectal cancer patients is compared with normal healthy controls, the expression level of these miRNA and accuracy (RUC) (namely raise or lower).

Fig. 3: the respective expression level and the ROC tracing analysis that describe the miRNA of two rises (hsa-miR-409-3p and has-miR-671-3p) in colorectal cancer progress, these cases are selected and are obtained from Dukes ' A, the grouping of B, C and D cancer.Data obtain from microarray respectively, and obtain after contrasting has-miR-1238 stdn with internal stability.Data show that the miRNA feature detection found by the present invention is to the colorectal cancer in Dukes ' A stage.

Fig. 4: describe the mankind miRNA according to particularly preferably comprising in expression characteristic in second aspect of the present invention, object is to distinguish colorectal cancer and normal healthy controls, hepatocellular carcinoma and lung cancer further.Show that colorectal cancer is compared with lung cancer with normal healthy controls, hepatocellular carcinoma equally, the expression level of these miRNA of patient and accuracy (AUC) (namely raise or lower).Primary combination (hsa20-miR-33b*/hsa-miR-196a) shows that its accuracy as the diagnosis biomarker distinguishing CRC and healthy individuals, hepatocellular carcinoma and lung cancer is 85% (AUC=0.860).

Fig. 5: describe two tumours and to be correlated with the ROC tracing analysis example (hsa-miR-93 and hsa-miR-25) of miRNA, these two miRNA measure colorectal cancer by quantitative RT-PCR method and normal healthy controls blood plasma obtains (0: healthy individuals; 1: colorectal cancer).Result shows that these miRNA are as hypersensitivity and the specificity of diagnosing biomarker.

detailed Description Of The Invention

The present invention is based on following unexpected discovery, namely colorectal cancer can reliably be identified with high accuracy and susceptibility by miRNA expression characteristic specific in blood plasma, and wherein said expression characteristic typically comprises as defined herein by the upper mankind miRNA lowered that is in harmonious proportion.More particularly, the HCC of described miRNA expression characteristic-can be detected under early stage morbid state by miRNA expression pattern overall in analysed for plasma and/or each miRNA expression level-make and differentiate healthy individuals, hepatocellular carcinoma and lung cancer.

The present invention of following illustration can suitably do not exist not in this article concrete disclose one or more element any, one or more restriction condition under implement.

The present invention also will be described with reference to accompanying drawing according to specific embodiment, but the present invention is not limited, and only limits by claims.Described accompanying drawing is only schematic, is considered to nonrestrictive.

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

Using indefinite article or definite article such as " one " or " one " when referring to singulative noun, time " described ", comprising the plural form of this noun, unless otherwise indicated.

Term " approximately " refers to that those skilled in the art understand the accuracy interval of the technique effect that still can ensure object feature in the present invention.This term ordinary representation depart from indicator value ± 10%, preferably ± 5%.

In addition, term first, second, third, (a), (b), (c) etc., in the specification and in the claims for element like region class, are not that description order or chronological order are necessary.The term should understanding so application is interchangeable in appropriate situations, and the embodiment that the present invention describes can be different from other sequential operation of described herein or illustration.

Term be defined in following use term further time provide.

Following term or definition only provide to understand the present invention.These definition should not be considered to have the scope being less than those skilled in the art and understanding.

An object of the present invention is by measuring multiple nucleic acids molecule in blood, for Colorectal Cancer Diagnosis, monitoring cancer therapy, and/or treatment colorectal cancer provides new method, often kind of nucleic acid molecule is all encoded microRNA (miRNA) sequence, one or more in wherein said multiple nucleic acids molecule are in plasma of colorectal cancer and healthy individuals, differential expression in hepatocellular carcinoma and lung cancer blood plasma, the nucleic acid molecule of one or more differential expressions wherein said represents expression of nucleic acid feature together, this expression of nucleic acid feature is the indication that colorectal cancer exists, expression of nucleic acid feature wherein comprises tumour correlated characteristic and plasma specific feature.

Term " Colon and rectum " herein refers to colon, rectum and/or appendix, i.e. whole large intestine.

Herein term " cancer " (also referred to as cancer) is often referred to the malignant neoplasm of any type, namely shows compared with unaffected (health) wild type control cells or has any morphology that the target cell that cancer feature is inclined to occurs and/or physiology changes (based on hereditary reprogrammed (genetic re-programming)).The example of this change can relate to cell size and shape (become large or diminish), cell proliferation (cell count increase), cytodifferentiation (physiological status change), apoptosis (apoptosis) or cell survival.Therefore, term " colorectal cancer " refers to the cancerous growths of colon, rectum and appendix.

Modal colorectal cancer (CRC) cell type is gland cancer, accounts for 95% greatly.The CRC of other types comprises especially lymphoma and squama cancer.

Colorectal cancer can according to Dukes phylogenetic systematics (Dukes, C.E. (1932) J.Pathol.Bacteriol.35,323-325).Comprise by stages following: Dukes A-tumour is confined to intestines wall; Dukes B-tumor invading is through intestines wall; Dukes C-tumour involves in lymphoglandula; Dukes D-tumour has distant metastasis.

Term " blood plasma " refers to the yellow liquid composition of blood herein, and wherein the hemocyte of whole blood can suspend usually.Account for greatly 55% of whole blood volume.The overwhelming majority is water (90% volume) and contains the albumen, glucose, Rh factor, mineral ion, hormone and the carbonic acid gas (blood plasma is the main medium of secretory product transport) that dissolve.Blood plasma is by centrifugal in centrifuges until prepare bottom blood cell sedimentation to centrifuge tube by fresh blood.Then blood plasma is purified and shifts.The density of blood plasma is approximately 1025kg/m3, or 1.025kg/l.Research now shows that miRNA is stable in blood plasma.Term " plasma sample " refers to the blood plasma obtained from detected individuality or normal healthy controls.

Term used herein " patient ", refers to the people that at least should be considered to suffer from hepatocellular carcinoma; Term used herein " target plasma " refers to the blood plasma obtained from patient; Term " healthy individuals " or " normal healthy controls " refer in particular to the healthy individuals without arbitrary cancer displays.And " contrast blood plasma " refers to the blood plasma obtained from these healthy individuals here.Such as, but in some application, when more different cancer types, these people have other cancer types, and these blood plasma obtained from these individualities are also refered in particular to as " contrast ".

Usually, plasma sample used derives from the biological sample of the research object being diagnosed as colorectal cancer.In addition, in order to more determine to obtain data from " contrast sample ", can collect from the research object of known morbid state.Biological sample can comprise bodily tissue and liquid, such as colorectal carcinoma, serum, hemocyte, sputum and urine.In addition, biological sample can obtain from having colorectal cancer feature or suspected case.In addition, if any being necessary, sample can obtain by purifying from the bodily tissue obtained and liquid, so with as biological sample.According to the present invention, the expression level of labeled nucleic acid molecule is measured by the biological sample that research object is originated and obtains.

Sample for detecting in vitro method of the present invention should be collected in clinical acceptable mode usually, preferably to protect the mode of nucleic acid (particularly RNA) or protein to collect.Sample to be analyzed blood typically.In addition, hepatic tissue and other type of sample also can use.Sample can merge especially after initial processing.But also can use the sample do not merged.

Term used herein " microRNA " (or " miRNA ") is its its ordinary meaning in this area (Bartel, D.P. (2004) Cell 23,281-292; He, L.and Hannon, G.J. (2004) Nat.Rev.Genet.5,522-531).Therefore, " microRNA " refers to the RNA molecule derived from genomic gene seat, and it is from the transcript processing that can form 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, such as 18,19,26 or 27 Nucleotide.

MiRNA encoding sequence has the potentiality of matching with flanking genomic sequence, within the RNA duplex making ripe miRNA be placed on non-fully pairing (herein also referred to as stem-ring or hairpin structure or pre-miRNA), described duplex is as the intermediate carrying out miRNA processing from longer precursor transcript.This processing occurs typically via the continuous action of be called Drosha and Dicer two species specific endonucleases.Drosha produces the miRNA precursor (herein also referred to as " pre-miRNA ") being typically folded into hair clip or stem-ring structure from primary transcript (herein also referred to as " pri-miRNA ").From this miRNA precursor, cut miRNA duplex by Dicer, it comprises ripe miRNA at the one arm of hair clip or stem-ring structure, comprises the sections (being commonly referred to miRNA*) of similar size in another arm.Then miRNA is directed to its said target mrna to play its function, and miRNA* is degraded.In addition, miRNA is typically derived from the genome segment different from the protein coding region of prediction.

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

Term used herein " nucleic acid molecule of coding microrna sequences " refers to any nucleic acid molecule of coding microRNA (miRNA).Therefore, this term not only refers to ripe miRNA, also refers to corresponding precursor miRNA as above and elementary miRNA transcript.In addition, the invention is not restricted to RNA molecule, also comprise the DNA molecular of corresponding coding microRNA, such as, by DNA molecular that reverse transcription miRNA sequence produces.The nucleic acid molecule of microrna sequences of the present invention of encoding typically is encoded single miRNA sequence (i.e. individual miRNA).Such as, but the also possibility two or more miRNA sequence of this nucleic acid molecule encoding (i.e. two or more miRNA), a transcription unit is included in the conventional sequence that regulates as the two or more miRNA sequences under promotor or transcription terminator control.

The term used herein nucleic acid molecule of microrna sequences " coding " is also understood to include " having phosphorothioate odn molecule " (i.e. nucleotide sequence (5 ' → 3 ') mate or corresponding to molecule of coded miRNA (5 ' → 3 ') sequence) and " antisense nucleic acid molecule " (namely nucleic acid array complementation is in coded miRNA (5 ' → 3 ') sequence or the molecule of reverse complementary sequence (3 ' → 5 ') in other words mating coded miRNA sequence).Term used herein " complementation " refers to that " antisense " sequence of nucleic acid molecules and corresponding " having justice " sequence of nucleic acid molecules (having the sequence being complementary to antisense sequences) form the ability of base pair, preferably Watson-Crick base pair.

Within the scope of the present invention, two nucleic acid molecule (namely " having justice " and " antisense " molecule) can complete complementary, and namely they are containing any base mispairing and/or Nucleotide that is extra or disappearance.Or two molecules comprise one or more base mispairing or different on their total nucleotide number (causing owing to adding or lacking).Preferably, " complementation " nucleic acid molecule comprises at least 10 continuous nucleotides showing complete complementary to the sequence be included in corresponding " having justice " nucleic acid molecule.

Therefore, the multiple nucleic acids molecule being included in the coding miRNA sequence in diagnostic kit of the present invention can comprise one or more " has phosphorothioate odn molecule " and/or one or more " antisense nucleic acid molecule ".Sometimes, diagnostic kit comprises one or more " has phosphorothioate odn molecule " (i.e. miRNA sequence itself), described molecule is considered to the entirety of the miRNA (i.e. molecule marker) constituting differential expression or at least sub-set, the miRNA of described differential expression is the indication that there is or occur particular condition tendency, is colorectal cancer herein.On the other hand, when diagnostic kit comprises one or more " antisense nucleic acid molecule " sequence of miRNA sequence complementation (namely with), described molecule can comprise probe molecule (for carrying out hybridization assays) and/or the Oligonucleolide primers (such as applying for reverse transcription or PCR) of one or more specific (complementation) miRNA sequence being suitable for detecting and/or in quantitative given sample.

The multiple nucleic acids molecule defined in the present invention 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, often kind of molecule encoding miRNA sequence.

Term used herein " differential expression " refers to that the expression level of specific miRNA in target plasma is change compared to normal healthy controls blood plasma, and it can be raise (namely miRNA concentration increases in target plasma) or lower (namely miRNA concentration reduces or disappears in target plasma).In other words, nucleic acid molecule is activated to than higher or lower level in contrast blood plasma in target plasma.

In the scope of the invention, nucleic acid molecule is considered to differential expression, if the corresponding expression level of this nucleic acid molecule in target cell and compared with 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 value of the latter 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 compared to wild type control cells, otherwise or expression level in target cell lower at least 0.7 times or at least 0.5 times.

Term used herein " expression level " refers to that specific miRNA sequence is from the transcribed degree of its genomic gene seat, i.e. the concentration of miRNA in one or more analyzed blood plasma.

As mentioned above, term " contrast blood plasma " typically refers to (health) blood plasma without HCC phenotypic characteristic.Such as, but in some applications, when comparing the blood plasma of the different cancer of display or precancerous condition, the blood plasma with more not serious genius morbi is typically considered to " contrast blood plasma ".

Standard program (the Sambrook set up well known in the art is typically followed in the determination of expression level, 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 Pro-cols in Molecular Biology.Wiley & Sons, Hoboken, NJ).Determine to carry out at rna level, such as, use miRNA specific probe to carry out Northern engram analysis, or such as carried out at DNA level by quantitative PCR or real time pcr after reverse transcription (and clone) RNA group.Any nucleic acid molecule comprising the above-mentioned microrna sequences of analysis of encoding " determined " in term used herein.But, because pri-miRNA and the re-mRNA transformation period is short, typically only measure the concentration of ripe miRNA.

In particular embodiments, the standard value of the expression level obtained in some independent measurements (such as two, three, five or ten measurements) of given sample and/or the some measurements in a group target plasma or contrast blood plasma is used to analyze.Standard value can obtain by any method known in the art.Such as, the scope of mean value ± 2SD (standard deviation) or mean value ± 3SD can be used as standard value.

Difference between the disease obtained or contrast plasma level can be normalized to the expression level of further contrast nucleic acid such as house-keeping gene, and the expression level of house-keeping gene is known not according to obtaining the morbid state of individuality of sample and different.The house-keeping gene of citing comprises beta-actin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein P1 etc.In preferred embodiments, contrasting nucleic acid is the known another kind of miRNA collecting stably express in the different non-cancer of sample and cancer (front) state.

But, replace measuring plasma sample expression level in any experiment, also can based on experimental evidence and/or prior art data definition one or more cutoff value for specified disease phenotype (i.e. morbid state).In this case, the grid expression level of plasma sample can measure with the contrast miRNA for normalized stably express.If the expression level of " normalization method " that calculates 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 " normalization method " that calculates is lower than the cutoff value of corresponding definition, then this discovery is the indication of down regulation of gene expression.

In the context of the present invention, term " qualification colorectal cancer and/or differentiation other types cancer " also comprises prediction and probability analysis (in " diagnosis " meaning).Composition disclosed herein and method are intended to clinical application, to determine form of therapy, comprise therapeutic intervention, and Case definition is as disease stage, and diseases monitoring and surveillance of disease.According to the present invention, the intermediate result of check object state can be provided for.This intermediate result can combine to help doctor, nurse or other practitioner to diagnose out this object to suffer from this disease with extraneous information.Or the present invention can be used for the cancer cells in detected object derived tissues, and provide useful information to doctor to diagnose.In addition, the present invention is also for distinguishing colorectal cancer and other types cancer comprises hepatocellular carcinoma and lung cancer.

In the present invention, the nucleic acid molecule of one or more differential expression identified represents a kind of expression of nucleic acid feature together, and this expression of nucleic acid feature is the indication existed by plasma sample qualification colorectal cancer.Term used herein " expression characteristic " refers to one group of nucleic acid molecule (such as miRNA), and wherein the expression level of each nucleic acid molecule is different between plasma of colorectal cancer and normal healthy controls.Herein, expression of nucleic acid feature also refers to a group echo and represents minimum object (difference) nucleic acid molecule, and often kind of nucleic acid molecule encoding can identify the miRNA sequence of individual phenotypic status.

In one aspect, the present invention relates to the diagnostic kit of the blood molecules mark for determining colorectal cancer, this test kit comprises multiple nucleic acids molecule, often kind of a kind of microRNA sequence of nucleic acid molecule encoding.In wherein said one or multiple nucleic acids molecule differential expression in target plasma and normal healthy controls group, and wherein the mark of differential expression derives from tumour is correlated with or plasma specific mark, and the nucleic acid molecule of this one or more differential expression represents the existence that an expression of nucleic acid mark sheet understands colorectal cancer together.

The expression of nucleic acid feature limited herein can comprise at least 35 nucleic acid molecule, preferably at least 12 kinds of nucleic acid molecule, and particularly preferably at least 6 nucleic acid molecule.

In preferred embodiments, nucleic acid molecule expression characteristic comprises the nucleic acid molecule of at least one coding miRNA sequence, one or more control group is compared in its expression in one or more blood plasma rise, and the nucleic acid molecule of at least one coding microRNA, one or more normal healthy controls is compared in its expression in one or more blood plasma downward.

Term used herein " derives from tumour " or " tumour source " refers to the feature of differential expression in Patients with Colorectal Cancer and contrast blood plasma, and in the Colorectal Carcinoma nucleus non-cancer tissue cell also differential expression.

Colorectal Carcinoma cell used herein, refers to and collects colorectal cancer cell from being diagnosed as colorectal cancer research object to cut out.Non-cancer tissue cell used herein typically refers to (health) wild-type cell not having cancer phenotypic characteristic.

Term used herein " plasma specific " refers to differential expression in Patients with Colorectal Cancer and contrast blood plasma, and does not have significant difference at colon cancer tissue cell and non-cancer tissue cell.

Usually, the nucleic acid molecule be included in expression of nucleic acid feature is human sequence (hereinafter referred to as " has " (homo sapiens (Homo sapiens))).

In a preferred embodiment of the invention, expression of nucleic acid feature comprises the relevant hsa-miR-409-3p (SEQ ID NO:1) of codes for tumor, hsa-miR-25 (SEQ ID NO:2), hsa-miR-93 (SEQ ID NO:3), hsa-miR-96 (SEQ ID NO:4), hsa-miR-301a (SEQ ID NO:5), hsa-miR-342-3p (SEQ ID NO:6), hsa-miR-19b (SEQ ID NO:7), hsa-miR-451 (SEQ ID NO:8), hsa-miR-486-5p (SEQ ID NO:9), hsa-miR-187* (SEQ IDNO:10), hsa-miR-92a (SEQ ID NO:11), hsa-miR-19a (SEQ ID NO:12), hsa-miR-20b (SEQ ID NO:13), hsa-miR-20a (SEQ ID NO:14), hsa-miR-139-3p (SEQ ID NO:15), hsamiR-107 (SEQ ID NO:16), hsa-miR-17 (SEQ ID NO:17), hsa-miR-140-3p (SEQ ID NO:18), hsa-miR-30e (SEQ ID NO:19), the expression of any one or the multiple nucleic acids molecule of hsa-miR-185 (SEQ ID NO:20), the hsa-mir-671-3p (SEQ ID NO:21) of coding plasma specific, hsa-mir-16-2* (SEQ ID NO:22), hsa-miR-30c-1* (SEQ ID NO:23), hsa-miR-548c-5p (SEQ ID NO:24), hsa-miR-16 (SEQ ID NO:25), hsa-miR-15a (SEQ ID NO:26), hsa-miR-425 (SEQ ID NO:27), hsa-let-7i (SEQ ID NO:28), hsa-miR-363 (SEQ ID NO:29), hsa-miR-15b (SEQ ID NO:30), hsa-miR-101 (SEQ ID NO:31), hsa-miR-190b (SEQ ID NO:32) and hsa-miR-130a (SEQ ID NO:33), hsa-miR-331-3p (SEQ IDNO:46), the expression of hsa-miR-345 (SEQ ID NO:47) any one or multiple nucleic acids molecule, the hsa-miR-1238 (SEQ ID NO:36) of encoding stable internal stability contrast and the nucleic acid molecule of hsa-miR-1228 (SEQID NO:37).

In order to the expression level normalization method by blood plasma nucleic acid molecule (nucleic acid molecule of the coding microrna sequences namely comprised in expression of nucleic acid feature), preferably can use miRNAhsa-miR-1238 (SEQID NO:44) and hsa-miR-1228 (SEQ ID NO:45), it is stably express in plasma of colorectal cancer.

The nucleotide sequence of above-mentioned miRNA lists in table 1.

table 1

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

Particularly preferably, compared with contrasting in blood plasma with at one or more, the expression of any one or the multiple nucleic acids molecule of hsa-miR-409-3p and hsa-mir-671-3p that encode in one or more target plasma described is raised, and the hsa-miR-25 that encodes, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsa-miR-486-5p, hsa-miR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa-miR-107, hsa-miR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185 ' hsa-mir-16-2*, hsa-miR-30c-1*, hsamiR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsa-miR-363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa-miR-130a, the expression of any one or the multiple nucleic acids molecule of hsa-miR-331-3p and hsa-miR-345 is lowered, hsa-miR-1238 and hsa-miR-1228 is unchanged.

As used herein, term " described multiple nucleic acids molecule one or more " and " nucleic acid molecule that any one or various human target cell are derivative " can relate to any subgroup of described multiple nucleic acids molecule, such as any one, any two, the nucleic acid molecule such as wantonly three kinds, wantonly four kinds, wantonly five kinds, wantonly six kinds, wantonly seven kinds, wantonly eight kinds, wantonly nine kinds, wantonly ten kinds, often kind of equal encoded packets of nucleic acid molecule is contained in the microrna sequences in described expression of nucleic acid feature.

In particularly preferred embodiments, adjust expression characteristic and comprise coding hsa-miR-409-3p/hsa-miR-16-2*, hsa-miR-409-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-548c-5p, hsa-miR-671-3p/hsa-miR-16-2*, hsa-miR-671-3p/hsa-miR-30c-1*, hsa-miR-671-3p/hsa-miR-342-3p, hsa-miR-671-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-301a, hsa-miR-671-3p/hsa-miR-345, hsa-miR-409-3p/hsa-miR-345, hsa-miR-409/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-25, the combination of any one or the multiple nucleic acids developed by molecule of hsa-miR-409-3p/hsa-miR-93 and hsa-miR-671-3p/hsa-miR-93.

Term used herein " Nucleic acid combinations " refers to applies at least two kinds of nucleic acid expression level on the whole.Preferably overallly can utilize change or calculation result relatively by a formula.

In preferred embodiments, expression of nucleic acid feature comprises coding: tumour correlated characteristic hsa-miR-409-3p (SEQ ID NO:1), hsa-miR-129-3p (SEQ ID NO:34), hsa-miR-33b* (SEQ ID NO:35), hsa-miR-7 (SEQ ID NO:36), hsa-miR-196b (SEQ ID NO:37), hsa-miR-93 (SEQ ID NO:3), hsa-miR-486-5p (SEQ ID NO:9), hsamiR-25 (SEQ ID NO:2), hsa-miR-92a (SEQ ID NO:11), hsa-miR-19b (SEQ ID NO:7), plasma specific feature hsa-miR-671-3p (SEQ ID NO:21), hsa-miR-16-2* (SEQ ID NO:22), hsa-miR-92b (SEQ ID NO:38), hsa-miR-129* (SEQID NO:39), hsa-miR-563 (SEQ ID NO:40), hsa-miR-602 (SEQ ID NO:41), hsamiR-1227 (SEQ ID NO:42), any one or the multiple nucleic acids molecule of hsa-miR-196a (SEQ ID NO:43) and internal stability contrast hsa-miR-1238 (SEQ ID NO:44) and hsa-miR-1228 (SEQ ID NO:45).

The nucleotide sequence of above-mentioned miRNA lists in table 2.

table 2

In a more preferred embodiment, with healthy individuals, hepatocellular carcinoma is compared with lung cancer, encode in one or more target plasma hsa-miR-409-3p, hsa-mir-671-3p, hsa-miR-33b*, hsa-miR-92b, hsa-miR-149, hsa-miR-129*, hsa-miR-563, hsa-miR-129-3p, hsa-miR-634, hsa-let-7b*, any one or the multiple nucleic acids developed by molecule of hsa-miR-602 and hsa-miR-1227 are raised, and the hsa-miR-16-2* that encodes, hsa-miR-7, hsa-miR-196a, hsamiR-196b, hsa-miR-486-5p, hsa-miR-93, hsa-miR-25, any one or the multiple nucleic acids developed by molecule of hsa-miR-92a and hsa-miR-19b are lowered, hsa-miR-1238 and hsa-miR-1228 does not change.

In a more preferred embodiment, expression of nucleic acid feature comprises coding hsa-miR-33b*/hsa20-miR-196a, hsa-miR-92b/hsa-miR-196a, hsa-miR-563/hsa-miR-196a, hsa-miR-1227/hsa-miR-196a, hsa-miR-129-3p/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-16-2*, hsa-miR-92b/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-196a, hsa-miR-1227/hsa-miR-16-2*, hsa-miR-129-3p/hsa-miR-196a, hsa-miR-602/hsa-miR-196a, hsa-miR-33b*/hsamiR-16-2*, hsa-miR-563/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-7, hsa-miR-33b*/hsa25-miR-7, hsa-miR-563/hsa-miR-7, hsa-miR-602/hsa-miR-16-2*, hsa-miR-129-3p/hsamiR-7, hsa-miR-92b/hsa-miR-7, hsa-miR-1227/hsa-miR-7, hsa-miR-33b*/hsa-miR-196b, hsa-miR-1227/hsa-miR-196b, any one or the multiple nucleic acids combination of hsa-miR-92b/hsa-miR-196b and hsa-miR-602/has-miR-7.

As used herein, term " expression of the nucleic acid molecule of change coding miRNA sequence " refers to and changes to cause the expression level of described molecule any manipulation of specific nucleic acid molecule, namely produces the different corresponding miRNA measured compared with the expression of " wild-type " (i.e. unaltered contrast).As used herein, term " different amount " had both comprised amount high compared with unaltered contrast, also comprised lower amount.In other words, handling as herein defined can be the expression (namely particularly transcribing) of raising (namely activating) or lowering (namely suppressing) nucleic acid molecule

In the present invention, the expression of one or more nucleic acid molecule of the coding microrna sequences comprised in expression of nucleic acid feature is modified by this way, and it is expressed and to be lowered by the expression of the nucleic acid molecule raised in one or more target plasma described and its expression is raised by the expression of the nucleic acid molecule lowered in one or more target plasma described thus.In other words, the modification of expression of the specific nucleic acid molecule of coding miRNA sequence with the generation of the antimode (anti-cyclical) with the regulating effect of described molecule in one or more cancer target plasma described, to disturb by " overactivity " of molecule that raise in one or more target plasma described and/or to recover by " defect is active " of the molecule lowered.

In a preferred embodiment of the inventive method, lower the expression of nucleic acid molecule and comprise coding is imported in patient body with by the nucleic acid molecule of the sequence of the microrna sequences complementation of nucleic acid molecule encoding lowered.

Term as used herein " in importing blood " refers to and makes one or more nucleic acid molecule shift any manipulation entered in blood.The example of this technology comprises injection well known in the art, digestion and other technologies.

Term as used herein " complementary sequence " refers to that " complementation " nucleic acid molecule (herein also referred to as " antisense nucleic acid molecule ") imported in one or more cell can form base pair, preferred Watson-Crick base pair with endogenous " having justice " nucleic acid molecule raised.

Two kinds of nucleic acid molecule (namely " having justice " and " antisense " molecule) can be complete complementaries, and namely it is containing any base mispairing and/or interpolation or deleted nucleotides.In other embodiments, these two kinds of molecules comprise one or more base mispairing or its total nucleotide number difference (owing to adding or lacking caused).In further embodiment, " complementation " nucleic acid molecule comprise one section with at least ten continuous nucleotides of sequence complete complementary of comprising in " having justice " nucleic acid molecule raised.

" complementation " nucleic acid molecule (i.e. the nucleic acid molecule of nucleotide sequence of coding and the microrna sequences complementation of the nucleic acid molecule encoding of downward) can be the nucleic acid molecule of the DNA-of natural generation or RNA molecule or the synthesis comprising one or more identical type or one or more dissimilar modified nucleotide in its sequence.

Such as, at least one ribonucleotide main chain unit and at least one deoxyribonucleotide main chain unit may be comprised by this nucleic acid molecule.In addition, described nucleic acid molecule can be 2 '-O-methyl group or 2 '-O-methoxy group (methylating also referred to as 2 '-O-) containing one or more RNA backbone modifications, it prevents nuclease degradation in the medium, and be importantly also prevent the kernel of RNA inducibility silencing complex nuclease to be separated, cause the irreversible suppression of miRNA.Another possible modification (its function equivalence methylates in 2 '-O-) comprises locked nucleic acid (LNA), the nucleic acid analog of representative containing one or more LNA nucleotide monomer, described monomer is simulated in sugared conformation at RNA has locking bifuran sugar unit (Orom, U.A.et al. (2006) Gene 372,137-141).

Be developed recently another kind of miRNA expression silencing gene.The chemical engineering oligonucleotide that these are called " antagomirs " is the RNA molecule (Krutzfeldt, J.et al. (2005) Nature 438,685-689) of strand 23 Nucleotide puted together with cholesterol.As this chemically modified oligonucleotide another select, create as RNA produce from transgenosis can at the microRNA inhibitor of cells.These competitive inhibitors being called " microRNA sponge (microRNA sponges) " are the transcripts of expressing from strong promoter, multiple series combination site (Ebert containing interested microRNA, M.S.et al. (2007) Nat.Methods 4,721-726).

In the particularly preferred embodiment of the inventive method, it is expressed and is selected from the microrna sequences as next group relative to expression characteristic by one or more nucleic acid molecule encoding lowered: hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsamiR-486-5p, hsa-miR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa-miR-107, hsa-miR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185, hsa-miR-16-2*, hsa-miR-30c-1*, hsa-miR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsa-miR-363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa-miR-130a, hsa-miR-7, hsa-miR-196b and hsa-miR-196a, may be the indication of colorectal cancer as mentioned above.

In another preferred embodiment of the inventive method, raise nucleic acid molecule and express to comprise coding is imported in blood by the nucleic acid molecule of the microrna sequences of nucleic acid molecule encoding raised.In other words, the rise of the expression of the nucleic acid molecule of coding miRNA sequence realizes by being imported in one or more cell described by another copy (namely other " having justice " nucleic acid molecule) of described miRNA sequence.Described " having justice " nucleic acid molecule imported in one or more target cell can comprise the modification identical with above-mentioned " antisense " nucleic acid molecule.

In the particularly preferred embodiment of the inventive method, it is expressed and is selected from the microrna sequences as next group relative to expression characteristic by one or more nucleic acid molecule encoding raised: hsa-miR-409-3p, hsa-miR-671-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-92b, hsa-miR-129*, hsa-miR-563, hsa-miR-602 and hsa-miR-1227 may be the indication of colorectal cancer as mentioned above.

Import in blood and operably can be connected described nucleotide sequence is expressed with adjustment sequence with " having justice " and/or " antisense " nucleic acid molecule modifying the expression of the nucleic acid molecule of the microrna sequences comprised in one or more coding nucleic acid expression characteristic.

In order to any potential association of miRNA differentiated in sample before illustrating carcinous or cancer, the preparation function analysis of the discriminating about the combinable mRNA target sequence of described miRNA can be carried out.Based on discovery miRNA both can participate in tumor suppressor also can participate in tumour occur (Esquela-Kerscher, A.and Slack, F.J (2006) are as above; Calin, G.A.and Croce, C.M. (2007) are as above; Blenkiron, C.and Miska, E.A. (2007) are as above), can infer that the mRNA target site of this miRNA comprises tumor suppressor gene and oncogene.

If nucleic acid molecule comprises containing about transcribing and/or the sequential element of translational regulation information, and this sequence " operably connects " in the nucleotide sequence of coded polypeptide, then claim this nucleic acid molecule for " energy express nucleic acid molecule " or can " nucleotide sequence be expressed ".Operably connect is wherein said adjustment sequential element with the sequence be expressed (and/or mutual sequence expressed) so that the mode of genetic expression can be made to carry out the connection be connected.

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

In addition, also can by such as there is the Nucleotide of modification and affecting (as mentioned above) in the expression of nucleic acid molecule as defined herein.Such as, locked nucleic acid (LNA) monomer be considered to by strengthen to the resistance of degraded and increased miRNA in body by the stable miRNA-target duplex structure for silencing activity key functional half-life (see such as Naguibneva, I.et al. (2006) Biomed.Pharmacother 60,633-638).

Therefore, the nucleic acid molecule of the present invention be imported in the blood provided can comprise adjustment sequence, and preferred promoter sequence, optionally also comprises transcription termination sequence.Described promotor can allow composing type or inducible gene expression.Suitable promoter comprises intestinal bacteria (E.coli) lacUV5 and tet (tsiklomitsin response) promotor, T7 promotor and SV40 promotor or CMV promoter.

Nucleic acid molecule of the present invention also can be included in carrier or other cloning vector as in plasmid, phagemid, phage, clay or artificial chromosome.In preferred embodiments, described nucleic acid molecule comprises in the carrier, is particularly included in expression vector.Can comprise except the nucleotide sequence of the genetic constructs that this expression vector defines as the present invention except above-mentioned adjustment sequence and coding and copy derived from the species with the host compatibility for expressing the selective marker can selecting phenotype with control sequence and the cell of giving transfection.Known in the art and commercially available many suitable carriers are as pSUPER and pSUPERIOR.

Within the scope of the present invention, suitable pharmaceutical cpd comprise be applicable to oral cavity, rectum, nasal cavity, overall (comprising cheek and lower jaw), peritonaeum with parenteral (comprising intramuscular, subcutaneous and vein) operation, or by sucking or being blown into enforcement.Operation can be local or system.Preferably, operate by oral cavity or Intravenous administration route realization.Formula can be packaged into different dose units.

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

Above-mentioned (" having justice " and " antisense ") nucleic acid molecule can be formulated as pharmaceutical composition (Gennaro by the use acceptable composition of pharmacology and the preparation method set up, 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) AGuide to Pharmaceutical Particulate Science.Interpharm/CRC, Boca Raton, FL; Niazi, S.K. (2004) Handbook of Pharmaceutical Manufacturing Formulations, CRC Press, Boca Raton, FL).

In order to pharmaceutical compositions, the inorganic of pharmaceutical inert or organic excipients (i.e. carrier) can be used.In order to preparation example is as pill, tablet, capsule or particle, such as lactose, talcum, stearic acid and salt thereof, fat, wax, solid or liquid polyol, natural oil or winterized stearin can be used.For the production of solution, suspension, milk sap, aerosol mixture or before the use reprovision be that the appropriate excipients of the powder of solution or aerosol mixture comprises water, alcohol, glycerine, polyvalent alcohol and suitable mixture thereof and vegetables oil.

Described pharmaceutical cpd also can contain additive, as filling agent, bonding agent, moistening agent, glidant, stablizer, sanitas, emulsifying agent, and other solvent or solubilizing agent or realize the material of storage effect.The latter can be regarded as and nucleic acid molecule can be mixed in slowly-releasing or sustained release or targeted delivery system as in liposome, nano particle and micro-capsule.

In order to the great majority tissue in target body, need clinical feasible nothing wound strategy so that this pharmaceutical composition is as defined herein oriented to cell.In the past, certain methods by the siRNA of Rational Dosage is entered in mouse and primate bodies to have obtained great treatment benefit through intravenous injection, and without obvious limiting toxicity.

A kind of method comprises passerby's chain of miRNA (miRNA* chain) and cholesterol or derivatives thereof/conjugate covalent coupling to promote by time at the absorption (Soutschek of the cell surface LDL receptors of expressing, J.et al. (2004) Nature 432,173-178).Or the oligonucleotide (LNA-antimiR) that the locked nucleic acid of unconjugated PBS-preparation is modified may be used for general conveying (Elmen, J.et al. (2008) Nature 452,896-899).The method of another kind of conveying miRNA comprises use polyoxyethylene glycol makes miRNA capsulation become specific liposome to reduce the absorption of scavenger cell and to strengthen cycling time.MiRNA is delivered to liver and (and does not arrive other organ (Zimmermann by these specific nucleic acid particle (stable nucleic acid-lipid particle or SNALP) effectively; described in T.S.et al. (2006) Nature 441,111-114)).In recent years, describe the agent delivery (Akinc of novel lipid sample delivery of molecules (synthesizing based on alkyl acrylate or alkyl-acrylamide and primary amine or the puting together addition of secondary amine) as RNAi therapeutical agent that a class is called lipidoids, A.et al. (2008) Nat.Biotechnol.26,561-569).

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

The present invention is further described by accompanying drawing and following embodiment, and described drawings and Examples, just in order to the object of illustration special embodiment of the present invention, should not be construed as the meaning limiting the scope of the invention by any way.

Embodiment

embodiment 1: sample collection and preparation

The normal Colorectal Carcinoma sample of 69 routine Colorectal Carcinomas and 69 example couplings obtains from operation.Operation sample collect time or collect after very fast in liquid nitrogen frozen section, Sample preservation is at-80 ° of C.

Patient information (age, sex, image data, treatment, other drug treatment situation, family history and similar information) derives from hospital's database, to make the different sample matches collected.Pathology are followed up a case by regular visits to, and (such as by the histologic analysis of Hematorylin and eosin stains (H & E)) is used to significantly determine given sample morbid state (i.e. contrast, precancer (such as Colon and rectum adenoma), primary malignant pathology (such as colorectal cancer)), and guarantees the consistent classification of sample.

Detection wind lidar is optionally carried out with specific isolation tumor cell group (about 200000 cells) to each cancer sample.In brief, transparent transfer film is applied to the surface of tissue slice or sample.Under the microscope, observe the thin tissue section be placed on slide glass, and identification of cell group is to be separated.When the cell selected is positioned at the center of field of view, activate the integrated microscope optics of near-infrared laser diode (near IR laser diode integral with the microscopeoptics).Pulse laser beam activates the spot (spot) on transfer film, makes the cytogamy of this film and selection below.Then the transfer film with the cell of combination is peeled off from described thin tissue section and (summarize see such as Emmert-Buck, M.R.et al. (1996) .Science 274,998-1001; Espina, V.etal. (2007) Expert Rev.Mol.Diagn.7,647-657).Substantially prepare freezing microtome section as manufacturers instructions and use laser capture microscope (Arcturus Veritas ^tMlaser Capture MicrodissectionInstrument (Molecular Devices, Inc., Sunnyvale, CA, USA) carries out catching step.

Use mirVana ^tMmiRNA separating kit (Ambion, Inc., Austin, TX, USA) is separated miRNA group according to manufacturers instructions.Concentration is quantitatively recorded by NanoDrop 1000 spectrophotometer.(NanoDrop Technologies,Waltham,MA)。The quality control of RNA uses RNA 6000Pico LabChip test kit to realize (Agilent Technologies, Santa Clara, CA) by 2100Bioanalyzer.

embodiment 2: the analysis of miRNA express spectra in tissue sample

Agilent miRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA) is used optionally to carry out qualitative analysis to the miRNA that (difference) in specific sample is expressed.Microarray comprises 723 mankind miRNA probes v.10.1 obtained from Sanger database.General RNA (100ng) derives from the colorectal cancer sample that each 138LCM-selects, and is used to by Cy3 totally as tag set.Microarray imaging results is obtained (PMT100, PMT5) by XDR scanning.Mark and hybridization realize according to Agilent miRNA microarray system step.

By the raw data normalization method (Agilent Technologies, Santa Clara, CA, USA) of applying Quantile method and use GeneSpringGX10 software known in the art will obtain for monochromatic (CY3) hybridization.Unpaired t-test (p value <0.01) is used to determine the miRNA of differential expression in colorectal cancer and coupling normal control tissue after measuring (F-test) through Fisher.

In order to each observed value implements the independent experiment for 138 tissue samples, and measure the mean value of the individual data that the representative of miRNA expression level obtains.

embodiment 3: the collection of plasma sample and preparation

Determine that the Main process steps of one or more expression of nucleic acid feature in colorectal cancer target plasma is shown in Fig. 1.

114 blood samples obtained from cancer patient and healthy individuals obtain in 2008 and 2009 from upper marine mountain and the collection of Huashan hospital.The essential characteristic diagram 3 of the blood sample used in the present invention.From patient obtain so sample is obtained by operation.Patient data (age, sex, image data, methods for the treatment of, other medical conditions, family history etc.) derives from hospital's database.The histopathologic classification of tumour is according to WHO staging system and is obtained by three pathologist's complete independentlies.

table 3: the essential characteristic of blood preparation

Full-length genome miRNA analyzes	Number
		Contrast
Normally	23
		Hepatocellular carcinoma	24
Lung cancer	36
		Intestinal cancer
Dukes ' A cancer	6
		Dukes ' B cancer	9
Dukes ' C cancer	9
		Dukes ' D cancer	7
Quantitative RT PCR analysis
		Normally	54
Intestinal cancer	54
		Sum	222

Peripheral blood (2ml) transfers to EDTA pipe.In two hours, it is centrifugal that EDTA pipe carries out 820g10min.Then 1-ml aliquot blood plasma transfers to 1.5-ml tubule and centrifugal 16,000g10min, to precipitate so residual cell debris.Then supernatant is transferred to new tubule and is stored in-80 DEG C.

General RNA also instructs extraction to obtain (Ambion, Austin, TX) according to businessman by use mirVana PARIS miRNA extraction agent box from blood plasma.Concentration is quantitatively obtained (NanoDrop Technologies, Waltham, MA) by NanoDrop 1000 spectrophotometer.RNA quality control is by 2100 Bioanalyzer and use RNA 6000 Pico LabChip test kit to be achieved (AgilentTechnologies, Santa Clara, CA).

embodiment 4: miRNA expression pattern analysis in plasma sample

MiRNAf difference in specific blood plasma sample) qualitative analysis optionally use AgilentmiRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA).Microarray comprises the probe of 723 mankind miRNA v.10.1 obtained from Sanger database.General RNA (100ng) derives from each 114 plasma sample, is used to by Cy3 totally as tag set.Microarray is affected result and is obtained (PMT100, PMT5) by XDR scanning.Mark and hybridization realize according to Agilent miRNA microarray system step.

For data analysis, monochromatic (CY3) is hybridized the raw data obtained and is contrasted (has-miR-1238) normalization method by the inside miRNA that utilization one is stable.Unpaired t-test is used to determine the miRNA of colorectal cancer and healthy individuals and/or colorectal cancer and the middle differential expression of contrast (healthy individuals, hepatocellular carcinoma and lung cancer) after measuring (F-test) through Fisher.

For determining that individual miRNA is as specificity and the susceptibility of diagnosing biomarker, MedCalc software has been used to individual miRNA relative healths individuality and/or contrast (healthy individuals, hepatocellular carcinoma and lung cancer) accepts operating characteristics (ROC) tracing analysis.The credibility interval of 95% is used to determine its meaning.

In order to assess a specific miRNA in early days liver cancer or transfer liver cancer in variant expression compared with healthy individuals or contrast, use following standard:

I) multiple of p value (likelihood value) <=0.01 changes >2

Ii) AUC (accuracy as diagnosis biomarker) AUC>0.700

When achieving two kinds of standards, miRNA is considered to differential expression in colorectal cancer is relative to healthy individuals and/or contrast.

In order to obtain each measuring result, independent experiment is implemented for 114 plasma samples, and the mean value of each data of miRNA expression level representative acquisition.

CRC relative healths contrasts the differential expression miRNA experimental data summary obtained and shows following table 4-6.The miRNA expressed with notable difference in blood plasma in the tissue that CRC shows relative to control tissue and normal healthy controls blood plasma enumerated by table 4.Table 5 summarizes the miRNA that Patients with Colorectal Cancer is only present in differential expression in blood plasma for healthy individuals.Table 6 lists best miRNA characteristics combination in CRC case blood plasma." t " refers to Colorectal Carcinoma on hurdle, and " n " refers to the normal control tissue of coupling, and " p " refers to patient, and " h " refers to normal healthy controls.Particularly preferred miRNA (SEQ IDNO: table 4 is shown in SEQ ID NO:8; SEQ ID NO:21 is shown in table 5 to SEQ ID NO:24) divide for adding blackboard.

table 4: the tumour in plasma of colorectal cancer is correlated with miRNA feature

table 5: the plasma specific miRNA feature in plasma of colorectal cancer

table 6: the combination miRNA feature in plasma of colorectal cancer

Preferred expression characteristic for distinguishing colorectal cancer and healthy individuals, hepatocellular carcinoma and lung cancer in second aspect is listed in following table 7-9.Table 7 is enumerated colorectal cancer tumour and to be correlated with miRNA feature; Table 8 shows plasma specific miRNA feature; Table 9 shows the best of breed of colorectal cancer miRNA feature, and best of breed (hsa-miR-33b*/hsa-miR-196a) is 85% as the accuracy of the diagnosis biomarker distinguishing CRC and healthy individuals, hepatocellular carcinoma and lung cancer." t " hurdle is Colorectal Carcinoma, and " n " hurdle is coupling normal control tissue, and " CRC " hurdle is plasma of colorectal cancer, and " contrast " hurdle is the blood plasma obtained from healthy individuals, hepatocellular carcinoma and lung cancer patient.Preferred miRNA (SEQ ID NO:1; SEQ ID NO:34-37; SEQ ID NO:3 is shown in table 7, and SEQ ID NO:21, SEQ ID NO:38-42 shows and table 8, and SEQ ID NO:35 and SEQ ID NO:43 is shown in table 9) divide for adding blackboard.

table 7: distinguish colorectal cancer and normal healthy controls, hepatocellular carcinoma miRNA feature relevant with the tumour of lung cancer

table 8: the plasma specific miRNA feature distinguishing colorectal cancer and normal healthy controls, hepatocellular carcinoma and lung cancer

table 9: the combination miRNA feature distinguishing colorectal cancer and normal healthy controls, hepatocellular carcinoma and lung cancer

embodiment 5: examining of microarray data

For the miRNA expression data that checking (and/or quantitative) obtains, use the real-time quantitative RT-PCR set up according to manufacturers instructions, it adopts TaqMan MicroRNA assay method (AppliedBiosystems, Foster City, CA, USA).In brief, reverse transcription (RT) uses Taqman microRNA RT test kit and illustrates according to the biosystem used and instructs to realize.10ng total serum IgE reverse transcription solution mixture is 15ul, comprises 1X reverse transcription buffering, 1X RT primer, 1nM dNTP, 4U RNase inhibitor and 50U MultiScribe reversed transcriptive enzyme.Then RT solution in PCR instrument device by hot program 16 ° of C, 30min; 42 ° of C, 30min; 85 ° of C, 5min operation (Thermal cycler alpha engine, Bio-rad).Quantitative PCR measures test kit by TaqMan universal PC R Master Mix test kit and TaqmanmicroRNA and completes according to the biosystem teachings used.2ulRT product P CR amplification condition is: 1XTaqMan universal PC R Master Mix, does not measure mix containing AmpErase UNG, 1X TaqMan MicroRNA.Each step translation experience three circulation.The operational condition of PCR in real time is Roch Light Cycling 480 instrument, and program starts to heat 96 ° of C, 5min; Then 95 ° of C, 15s experience 45 or 50 circulations; 60 ° of C, 60s.Cp value by second deriving method of LC480 software according to obtaining.Then miRNA is by standard samples CP value absolute quantitation, and the CP value of each miRNA normalizes to internal stability contrast has-miR-1228.

The platform comparative experiments data of 8 miRNA combination and 7 miRNAS obtained from 54 routine healthy individuals and 54 routine plasma of colorectal cancer samples list in table 10.Generally speaking, the array fold difference of patient and normal healthy controls is greater than the result of quantitative RTPCR.The regulation and control (namely raise or lower) that array result obtains are large with quantitative RT-PCR result dependency.Result shows that the miRNA characteristic reliability found by Agilent miRNA microarray is large.

table 10: the platform of Patients with Colorectal Cancer blood plasma and healthy individuals blood plasma express spectra compares

The result obtained shows that colorectal cancer miRNA expresses the regulation and control of overall high specific.Therefore, miRNA subset concrete herein represents unique miRNA expression characteristic respectively, because Colon and rectum express spectra not only can determine that cancer state also can be distinguished with hepatocellular carcinoma and lung cancer.

MiRNA expression characteristic of the present invention be defined as blood screening, diagnosis and detection colorectal cancer provides a unique molecule marker.In addition, expression characteristic can be used to therapeutic response and the guiding treatment strategy of monitoring Patients with Colorectal Cancer.In addition, expression characteristic also can be used to development anti-colorectal carcinoma medicine.

This citing describe the present invention can suitably do not exist herein special disclose any element, restriction condition under carry out.Therefore, such as term " comprises ", " comprising " " to contain " etc. and should have broad sense and unrestricted.In addition; the term applied herein and express for describing the present invention and unrestricted meaning; and do not use these terms and express and get rid of any characteristic sum shown in it and describe or the meaning of Equivalent of its part, but should recognize can carry out various amendment in the scope of the invention of request protection.Therefore, although should understand the special announcement carried out the present invention by embodiment and optional feature, those skilled in the art can modify to the present invention and change, and this amendment and change are thought within the scope of the invention.

Describe the present invention herein extensive and upperly.The upper set of each narrower subordinate concept and Asia fallen within the scope of upper description also constitutes a part of the present invention.This comprises uses conditioned disjunction from the negative restriction of any theme of upper middle removing to upper description of the present invention, and no matter whether removed theme is quoted from this article especially.

Other embodiment is in following right.In addition, when feature of the present invention or all respects Ma Kushi prescription formula describe, those skilled in the art can recognize that the present invention is also described with each member any of Ma Kushi group or member's subgroup mode.

Claims

1., for differentiating that one or more shows the diagnostic kit of the blood Middle molecule marker of the target plasma of colorectal cancer, described test kit comprises the probe molecule of multiple nucleic acids molecule, often kind of nucleic acid molecule encoding microrna sequences,

One or more differential expression in target plasma and in one or more contrast blood plasma of wherein said multiple nucleic acids molecule,

The nucleic acid molecule of one or more differential expression wherein said represents expression of nucleic acid feature together, described expression of nucleic acid feature is the indication that colorectal cancer exists, wherein said expression of nucleic acid feature comprises coding hsa-miR-409-3p/hsa-miR-16-2*, hsa-miR-409-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-548c-5p, hsa-miR-671-3p/hsa-miR-16-2*, hsa-miR-671-3p/hsa-miR-30c-1*, hsa-miR-671-3p/hsa-miR-342-3p, hsa-miR-671-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-301a, hsa-miR-671-3p/hsa-miR-345, hsa-miR-409-3p/hsa-miR-345, hsa-miR-409/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-25, any one or the multiple nucleic acids molecular combinations of hsa-miR-409-3p/hsa-miR-93 and hsa-miR-671-3p/hsa-miR-93.

2. the test kit of claim 1, wherein said expression of nucleic acid feature can comprise at least 35 kinds of nucleic acid molecule.

3. the test kit of claim 1, wherein said expression of nucleic acid feature can comprise at least 12 kinds of nucleic acid molecule.

4. the test kit of claim 1, wherein said expression of nucleic acid feature can comprise at least 6 kinds of nucleic acid molecule.

5. the test kit of claim 1, wherein compared with one or more normal healthy controls, in one or more target plasma described: the expression of any one or the multiple nucleic acids molecule of coding hsa-miR-409-3p and hsa-mir-671-3p is raised; And any one or the multiple nucleic acids developed by molecule of encode hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-mir-16-2*, hsa-miR-30c-1*, hsa-miR-548c-5p, has-miR-331-3p and hsa-miR-345 are lowered.

6. the test kit of claim 1, is further used for colorectal cancer and healthy individuals, hepatocellular carcinoma and lung cancer to differentiate.

7. the test kit of claim 6, wherein said expression of nucleic acid feature can comprise at least 23 kinds of nucleic acid molecule.

8. the test kit of claim 6, wherein said expression of nucleic acid feature can comprise at least 18 kinds of nucleic acid molecule.

9. the test kit of claim 6, wherein said expression of nucleic acid feature can comprise at least 6 kinds of nucleic acid molecule.

10. the test kit of claim 6, wherein said expression of nucleic acid feature comprises any one or Multi-encoding: tumour correlated characteristic hsa-miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-7, hsa-miR-196b, hsa-miR-93, hsa-miR-486-5p, hsa-miR-25, hsa-miR-92a, hsamiR-19b; The nucleic acid molecule of plasma specific feature hsa-miR-671-3p and hsa-miR-16-2*, has-miR-92b, hsa-miR-129*, hsa-miR-563, hsa-miR-602, hsa-miR-1227, hsa-miR-196a and internal stability contrast hsa-miR-1238 and hsa-miR-1228.

The test kit of 11. claims 10, wherein compare with lung cancer with one or more healthy individuals, hepatocellular carcinoma, in one or more target plasma described, any one or the multiple nucleic acids developed by molecule of coding hsa-miR-409-3p, hsa-mir-671-3p, hsa-miR-33b*, hsa-miR-92b, hsa-miR-149, hsa-miR-129*, hsa-miR-563, hsa-miR-129-3p, hsa-miR-634, hsa-let-7b*, hsa-miR-602 and hsa-miR-1227 are raised; And any one or the multiple nucleic acids developed by molecule of encode hsa-miR-16-2*, hsa-miR-7, hsa-miR-196a, hsa-miR-196b, hsa-miR-486-5p, hsa-miR-93, hsa-miR-25, hsa-miR-92a and hsa-miR-19b are lowered; Hsa-miR-1238 and hsa-miR-1228 does not change.

The test kit of 12. any one of claim 6-11, wherein said expression of nucleic acid feature comprises coding: hsa-miR-33b*/hsa-miR-196a, hsa-miR-92b/hsa-miR-196a, hsa-miR-563/hsa-miR-196a, hsa-miR-1227/hsa-miR-196a, hsa-miR-129-3p/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-16-2*, hsa-miR-92b/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-196a, hsa-miR-1227/hsa-miR-16-2*, hsa-miR-129-3p/hsa-miR-196a, hsa-miR-602/hsa-miR-196a, hsa-miR-33b*/hsa-miR-16-2*, hsa-miR-563/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-7, hsa-miR-33b*/hsa-miR-7, hsa-miR-563/hsa-miR-7, hsa-miR-602/hsa-miR-16-2*, hsa-miR-129-3p/hsa-miR-7, hsa-miR-92b/hsamiR-7, hsa-miR-1227/hsa-miR-7, hsa-miR-33b*/hsa-miR-196b, hsa-miR-1227/hsa-miR-196b, any one or the multiple nucleic acids combination of hsa-miR-92b/hsa-miR-196b and hsa-miR-602/hsa-miR-7.

The test kit of 13. any one of claim 6-12 is for the preparation of the purposes differentiating by the following method to show in the composition of one or more target plasma of colorectal cancer, and described method comprises:

A () determines the expression level of multiple nucleic acids molecule in one or more target plasma described, often kind of nucleic acid molecule encoding microrna sequences;

B () determines the expression level of described multiple nucleic acids molecule in one or more normal healthy controls blood plasma; And

C respective expression level that () is obtained in step (a) and (b) by contrast, one or more nucleic acid molecule of differential expression in described target plasma and contrast blood plasma is identified from described multiple nucleic acids molecule, one or more nucleic acid molecule of wherein said differential expression represent together as any one of claim 1-12 the expression of nucleic acid feature that defines, described expression of nucleic acid feature is the indication that colorectal cancer exists.

The purposes of 14. claims 13, wherein said method is further used for colorectal cancer and healthy individuals, hepatocellular carcinoma and lung cancer to differentiate.

15. the test kit of any one of claim 1-12 is for the preparation of the purposes of monitoring by the following method in the composition for the treatment of of colorectal cancer, described method comprises:

A () differentiates expression of nucleic acid feature by using method defined herein in one or more target plasma; And

B () monitors the expression of one or more nucleic acid molecule of the coding microrna sequences that described expression of nucleic acid feature comprises in blood, described monitoring is carried out in such a way, namely the expression in its blood plasma is lowered after the treatment by the expression of the nucleic acid molecule raised before the treatment, and the expression in its blood plasma is raised after the treatment by the expression of the nucleic acid molecule lowered before the treatment.

The test kit of 16. any one of claim 1-12 is for the preparation of the purposes in the test kit of prevention or treatment colorectal cancer by the following method, and described method comprises:

A () differentiates expression of nucleic acid feature in blood by using the method described in claim 13 or 14; And

B () changes the expression of one or more nucleic acid molecule of the coding microrna sequences that described expression of nucleic acid feature comprises in blood, described change is carried out in such a way, namely its expression is lowered by the expression of the nucleic acid molecule raised in blood, and its expression is raised by the expression of the nucleic acid molecule lowered in blood.