CN102892898A

CN102892898A - Diagnostic kit including micro-rna biomarkers and used for diagnosis of hepatocellular carcinoma, and method

Info

Publication number: CN102892898A
Application number: CN2010800648029A
Authority: CN
Inventors: 吴莹; 朱虹光; 高雪; 卢韶华; 李兆勇; 任一萍; 李健
Original assignee: Fudan University
Current assignee: SHANGHAI LABWAY CLINICAL LABORATORY Co.,Ltd.
Priority date: 2009-12-24
Filing date: 2010-12-24
Publication date: 2013-01-23
Anticipated expiration: 2030-12-24
Also published as: CN102892898B

Abstract

The invention provides micro-RNA biomarkers and a diagnostic kit including DNA molecules of the micro-RNA biomarkers of a plurality of codes. The diagnostic kit is used for identifying one or more target plasmas expressing hepatocellular carcinoma. The invention also provides a method for diagnosis and treatment of the hepatocellular carcinoma, and a medicinal composite for prevention and treatment of the hepatocellular carcinoma.

Description

The diagnostic kit that contains the microRNA biomarker and the method that are used for diagnosis of hepatoma

Invention field

The present invention relates to composition and method for microRNA (microRNA) expression pattern analysis of hepatocellular carcinoma (hepatocellular cancer) blood plasma.

Background of invention

Hepatocellular carcinoma (being also referred to as liver cell cancer (hepatocellular carcinoma, HCC)) is one of modal solid malignant in the world wide.Its Main Tissues that has represented liver cancer is learned type, may account for the 70%-85% of all liver cancer cases.There is about 500,000 new cases every year in the world, and the death of almost identical number, this reflects this disease is lacked effective early detection and treats selection (Thorgeirsson, S.S.and Grisham, J.W. (2002) Nat.Genet.31,339-346; Parkin, D.M.et al. (2005) CA Cancer J.Clin.55,74-108).

Therefore hepatocellular carcinoma is the cancer of a class poor prognosis.Patient's prognosis is depended on when cancer is made a definite diagnosis by stages.5 years survival rate＜5% that patients with hepatocellular carcinoma is performed the operation, and 5 years survival rates of postoperative are 60%-70%.Row excision when tumor size＜2cm, survival rate can reach 86% in 5 years.(but tumor size＜5cm) is gone 3 years survival rates 17%-21% only of any curer to the early hepatocarcinoma patient.This show cancer early find for the treatment and survival of patients very important.(Tang,Z.Y.(2001)World?J?Gastroenterol?7,445-454;Chambers,A.F.et?al.(2002)Nat?Rev?Cancer?2,563-572;Mola-Kuba?D.et?al.(2006)Annals?of?Hepatology?5,16-24).

Although the morning of liver cell tumor, discovery and excision significantly improved patient's survival rate in recent years, most of tumours can not be to find in the non-fatal stage in its emergence period.Yet only the patients with hepatocellular carcinoma of about 10%-20% meets surgical conditions at present, and the surgical condition of described hepatocellular carcinoma is to determine according to parameters such as relatively normal liver function and accessible tumour infringements by available different clinical staging systems.In addition, the patient who carries out surgical blanking has high-frequency transfer/recurrence usually, and postoperative 5 annual survival rates only are 30%-40%.Liver cancer make a definite diagnosis common dependence Histological Evidence.Tissues sampled is by puncture needle sucking-off or biopsy.Yet some liver cancer well differentiateds that is to say that they are comprised of the liver cell of the maturation of almost completely growing, so these liver cancer tissues exactly like non-cancer hepatic tissue at microscopically.And not all Pathology Doctors ' is all trained to such an extent that can distinguish technicality between WD liver cancer and the normal liver tissue.Also having some Pathology Doctors 's can be liver gland cancer with the hepatocellular carcinoma mistaken diagnosis.Gland cancer is another type of liver cancer, and it generally originates from outside the liver.The more important thing is that its treatment of adenocarcinoma metastatic is completely different with primary hepatocarcinoma.Therefore these tumours that show as one type hepatocellular carcinoma the patient with it of early diagnosis are desirable for distinguishing dissimilar tumours and instructing different treatment plans, thereby can significantly improve long-term survival.

Hepatic tissue puncture or the modal danger of biopsy are hemorrhage, especially because liver cancer is the tumour (holding many blood vessels) that is rich in vascular.There are many cases may there is no need to carry out histodiagnosis by puncture or biopsy.Alfafoetoprotein (AFP) significantly improves in Hazard Factor (for example liver cirrhosis, chronic hepatitis B or chronic hepatitis C) that liver cancer occurs and the blood if patient has, and the doctor almost can just can not determine that liver cancer has occured this patient by biopsy.At present, (the a-alpha-fetoprotein AFP) is generally used for early detection hepatocellular carcinoma (Mizejewski, G.J. (2003) Expert Rev.Anticancer Ther.2,709-735 a kind of serum marker only; Paul, S.B.et al. (2007) Oncology 72, Suppl.1,117-123).Have low specificity yet this is single labelled, and usually because false positive results but inappropriate.The Serum AFP check only can be easy to detect liver cell tumor in 60% patient.On the other hand, in a large amount of liver cirrhosis patients, AFP also can raise when not having cancerous state.Therefore, we still need to determine some alternative molecule markers and develop some responsive hematologies and detect early discovery and differential diagnosis hepatocellular carcinoma.

A kind of approach that addresses this problem can be based on some little modulability RNA molecules, particularly based on microRNA (miRNA), they are little non-coding RNAs of the endogenous expression of a class evolution conservative, and size is 20-25 Nucleotide (nt), can mediate the expression of said target mrna.Since they were found just to be considered in cell development, differentiation, proliferation and apoptosis critical function is arranged before about 10 years.(Bartel,D.P.(2004)Cell?116,281-297,Ambros,V.(2004)Nature?431,350-355;He,L.et?al.(2004)Nat?Rev?Genet?5,522-531)。And miRNA has superiority than mRNA on as cancer biomarkers because they external very stable and also in vivo the life-span long.(Lu,J.et?al.,(2005)Nature?435,834-838;Lim,L.P.et?al.,(2005)Nature?433,769-773).

MiRNA is from the primary transcription deposits yields, and primary transcript is processed as stem-ring structure precursor (pre-miRNA) by RNase III Drosha.After transporte to cells matter, another kind is called the ring of the RNase III cutting pre-miRNA hair clip of Dicer and lacks double-stranded (ds) RNA to form, and wherein a chain mixes in the miRNA-protein (miRNP) as ripe miRNA.MiRNA instructs miRNP to arrive their said target mrna, and they bring into play function (Bartel, D.P. (2004) Cell 23,281-292 at this; He, L.and Hannon, G.J. (2004) Nat.Rev.Genet.5,522-531).

According to the complementary degree between miRNA and its target, miRNA can instruct different regulate processes.With the highly complementary said target mrna of miRNA by disturbing (RNAi) identical mechanism by special cutting with RNA.Therefore, in this case, the function of miRNA is as short interfering rna (siRNA).Guided with the lower said target mrna of miRNA complementarity and to enter cell degradation approach or being translated property and check and do not affect the mRNA level.But the mechanism of translation how miRNA checks their said target mrna still has arguement.

High-throughout miRNA quantitative technique, miRNA biochip technology for example, the real-time TaqMan miRNA based on RT-PCR analyzes, for whole cancer gene group miRNA profile research in the whole world provides powerful instrument.Obtainable available data shows that the dysregulation (dysregulation) that miRNA expresses may be relevant with generation and/or the development of some types of cancer.For example, shown two kinds of miRNA, miR-15 and miR-16-1 are positioned on the genetic loci of disappearance in the chronic lymphatic leukemia (CLL), and find that two kinds of miRNA genes are lacked or reduce in about 70% CLL patient.In addition, in the colorectum tumorigenesis, observe the downward modulation of miR-143 and miR-145, and the expression of miRNA let-7 often reduces (Michael, M.Z.et al. (2003) Mol.Cancer Res.1,882-891 in lung cancer; Mayr, C.et al. (2007) Science 315,1576-1579).In fact, cancer in expressing based on miRNA is relevant change and miRNA be usually located at the observation supposition miRNA of the genome area that participates in cancer may be both as tumor suppressor gene also as oncogene (Esquela-Kerscher, A.and Slack, F.J (2006) Nat.Rev.Cancer 6,259-269; Calin, G.A.and Croce, C.M. (2007) J.Clin.Invest.117,2059-2066; Blenkiron, C.and Miska, E.A. (2007) Hum.Mol.Genet.16, R106-R113).Point out the miRNA of unconventionality expression in these human cancer tissues to give prominence to them as the potential of the biomarker of diagnosis and prognosis.

Express spectra (Murakami, Y.et al. (2006) Oncogene 25, the 2537-2545 of the miRNA in the human hepatocellular carcinoma reported in some researchs; Li, W.et al. (2008) Int J Cancer 123,1616-1622; Huang, Y.S.et al. (2008) Hepatology 23,87-94; Ladeiro, Y.et al. (2008) Hepatology 47,1955-1963; Jiang, J.et al. (2008) Clin Cancer Res 14,419-427).In addition, these researchs demonstrate, compare with nonmalignant liver cell or tissue, and in pernicious cell or tissue, specific miRNA unconventionality expression.Therefore, this type of miRNA can provide relating to the understanding of the cell processes in pernicious transformation and the progress.

In many possible sample types, blood is considered to the optimal high-risk individuality of monitoring, and the early discovery of leading, morning are diagnosed, monitor and effectively treat cancer, because blood sample is easy to the method collection with Wicresoft.Be proved miRNA that tumour derives and be present in human plasma or the serum with the form of quite stable, not affected by endogenous RNA enzymic activity.These levels that are present in the miRNA that is derived from tumour in serum or the blood plasma are enough as the biomarker of cancer detection.And the miRNA in blood plasma or the serum has strong dependency, shows no matter be that blood plasma or serum sample all are suitable for clinical application miRNA as the biomarker of cancer diagnosis.(Mitchell,P.S.et?al.(2008)Proc?Natl?Acad?Sci?USA?105,10513–10518;Gilad,S.et?al.(2008)PLoS?ONE?3,e3148;Chen,X.et?al.(2008)Cell?Res?18,997-1006)。So far, also do not determine in the blood plasma of patients with hepatocellular carcinoma or serum based on the miRNA biomarker of blood sample.

Therefore, urgent need is based on the diagnostic flag of blood, the diagnostic flag of " expression characteristic (expression signature) " or " molecule footprint (molecular footprint) " form particularly so that can be fast, reliably with save into local diagnosing hepatocellular carcinoma.And still needing consistent method to come screening early hepatocyte cancer (early stage-HCC) in high-risk individuals, differential diagnosis HCC early finds liver cancer recurrence, and/or the monitoring liver cancer treatment.

Purpose of the invention and overview

The purpose of this invention is to provide for diagnosing hepatocellular carcinoma, the treatment of monitoring cancer, and/or the novel method for the treatment of cancer, wherein by measuring the multiple nucleic acids molecule in the blood, every kind of nucleic acid molecule microRNA (miRNA) sequence of all encoding, one or more of wherein said multiple nucleic acids molecule in the blood plasma of hepatocellular carcinoma, compare with normal healthy controls by analysis and/or and healthy individual, colorectal cancer is compared differential expression with lung cancer, the nucleic acid molecule of wherein said one or more differential expression represents the expression of nucleic acid feature together, the existence of this expression of nucleic acid feature indication hepatocellular carcinoma, wherein said expression of nucleic acid feature comprises Tumor-assaciated feature (tumor-related signature) and plasma specific feature (plasma-specific signature).

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

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

In first aspect, the present invention relates to the diagnostic kit for the blood molecular marked compound of differentiating the target blood plasma that one or more shows hepatocellular carcinoma, described test kit comprises the multiple nucleic acids molecule, every kind of nucleic acid molecule encoding microrna sequences, one or more of wherein said multiple nucleic acids molecule be differential expression in target blood plasma and in one or more contrast blood plasma, the nucleic acid molecule of wherein said one or more differential expression comes from Tumor-assaciated or plasma specific feature, the nucleic acid molecule of wherein said one or more differential expression represents the expression of nucleic acid feature together, the existence of described expression of nucleic acid feature indication hepatocellular carcinoma.

The expression of nucleic acid feature that this paper limits can comprise at least three ten two kinds of nucleic acid molecule, preferably at least ten two kinds of nucleic acid molecule, particularly preferably at least six kinds of nucleic acid molecule.

In preferred embodiments, described expression of nucleic acid feature comprises the nucleic acid molecule of at least a coding microrna sequences, and it is expressed in one or more target blood plasma to compare with one or more normal healthy controls and is raised; And comprising the nucleic acid molecule of at least a coding microrna sequences, it is expressed in one or more target blood plasma to compare with one or more normal healthy controls and is reduced.

In preferred embodiments, described expression of nucleic acid feature comprises codes for tumor correlated characteristic hsa-miR-122, hsa-miR-199b-3p, hsa-miR-192, hsa-miR-21, hsa-miR-139-3p, hsa-miR-10a, hsa-miR-103, hsa-miR-181d, hsa-miR-125b-2*, a-miR-125a-3p; Plasma specific feature hsa-miR-34a, hsa-miR-136, hsa-miR-151-5p, hsa-miR-193b*, hsa-miR-124, hsa-miR-936, hsa-miR-198, hsa-miR-149*, hsa-miR-138, hsa-miR-601, hsa-miR-769-3p, hsa-miR-513c, hsa-miR-525-5p, hsa-miR-654-5p, hsa-miR-518c*, hsa-miR-500, hsa-miR-181c*, hsa-miR-1226*, hsa-miR-202, any one of the contrast hsa-miR-1238 of hsa-miR-629* and internal stability and hsa-miR-1228 or multiple nucleic acids molecule.

Particularly preferably, compare with one or more normal healthy controls, in described one or more target blood plasma: the expression of any one of coding hsa-miR-122, hsa-miR-199b-3p, hsa-miR-192, hsa-miR-21, hsa-miR-10a, hsa-miR-103, hsa-miR-34a, hsa-miR-136, hsa-miR-151-5p or multiple nucleic acids molecule is raised; Coding hsa-miR-139-3p, hsa-miR-193b*, hsa-miR-124, hsa-miR-181d, hsa-miR-125b-2*, hsa-miR-125a-3p, hsa-miR-936, hsa-miR-198, hsa-miR-149*, hsa-miR-138, hsa-miR-601, hsa-miR-769-3p, hsa-miR-513c, hsa-miR-525-5p, hsa-miR-654-5p, hsa-miR-518c*, hsa-miR-500, hsa-miR-181c*, hsa-miR-1226*, hsa-miR-202, any one of hsa-miR-629* or multiple nucleic acids developed by molecule are reduced; Hsa-miR-1238 and hsa-miR-1228 do not change.

In a more preferred embodiment, the expression of nucleic acid feature comprises any one or the multiple nucleic acids molecule of codes for tumor correlated characteristic hsa-miR-122, hsa-miR-199b-3p, hsa-miR-192, hsa-miR-21, hsa-miR-139-3p, hsa-miR-10a, hsa-miR-103 and coding plasma specific feature hsa-miR-34a, hsa-miR-136, hsa-miR-151-5p, hsa-miR-193b*, hsa-miR-124.

Particularly preferably, compare with one or more normal healthy controls, in one or more target blood plasma: any one or more nucleic acid molecule of coding hsa-miR-122, hsa-miR-199b-3p, hsa-miR-192, hsa-miR-21, hsa-miR-10a, hsa-miR-103, hsa-miR-34a, hsa-miR-136, hsa-miR-151-5p is expressed and is raised; And any one or the multiple nucleic acids developed by molecule of coding hsa-miR-139-3p, hsa-miR-193b*, hsa-miR-124 are reduced.

In another preferred embodiment, the expression of nucleic acid feature comprises any one or the multiple nucleic acids molecule of codes for tumor correlated characteristic hsa-miR-122, hsa-miR-199b-3p, hsa-miR-192, hsa-miR-21, hsa-miR-139-3p and plasma specific feature hsa-miR-34a.

Particularly preferably, compare with one or more normal healthy controls, in one or more target blood plasma: the expression of any one of coding hsa-miR-122, hsa-miR-199b-3p, hsa-miR-192, hsa-miR-21, hsa-miR-34a or multiple nucleic acids molecule is raised, and the expression of any one or the multiple nucleic acids molecule of coding hsa-miR-139-3p is reduced.

In particularly preferred embodiments, the expression of nucleic acid feature comprises coding hsa-miR-34a/hsa-miR-193b*, hsa-miR-21/hsa-miR-936, hsa-miR-192/hsa-miR-124, hsa-miR-122/hsa-miR-193b*, hsa-miR-34a/hsa-miR-138, hsa-miR-34a/hsa-miR-198, hsa-miR-122/hsa-miR-124, hsa-miR-103/hsa-miR-139-3p, hsa-miR-192/hsa-miR-193b*, hsa-miR-122/hsa-miR-138, hsa-miR-34a/hsa-miR-139-3p, hsa-miR-122/hsa-miR-198, hsa-miR-122/hsa-miR-769-3p, hsa-miR-103/hsa-miR-193b*, hsa-miR-34a/hsa-miR-124, hsa-miR-192/hsa-miR-139-3p, hsa-miR-34a/hsa-miR-769-3p, hsa-miR-192/hsa-miR-936, hsa-miR-10a/hsa-miR-193b*, hsa-miR-122/hsa-miR-601, hsa-miR-21/hsa-miR-769-3p, hsa-miR-199b-3p/hsa-miR-193b*, hsa-miR-103/hsa-miR-138, hsa-miR-103/hsa-miR-198, hsa-miR-199b-3p/hsa-miR-139-3p, the combination of any one of hsa-miR-21/hsa-miR-139-3p and hsa-miR-21/hsa-miR-193b* or multiple nucleic acids molecule.

In second aspect, the present invention relates to for the diagnostic kit that differentiates with hepatocellular carcinoma and healthy individual, colorectal cancer and lung cancer.Described test kit comprises the multiple nucleic acids molecule, every kind of nucleic acid molecule encoding microrna sequences, one or more of wherein said multiple nucleic acids molecule be differential expression in target blood plasma and in one or more healthy individual, colorectal cancer and lung cancer, and the nucleic acid molecule of wherein said one or more differential expression represents the expression of nucleic acid feature together, the existence of described expression of nucleic acid feature indication hepatocellular carcinoma.

The expression of nucleic acid feature that this paper limits can comprise at least ten six kinds of nucleic acid molecule, preferably at least six kinds of nucleic acid molecule.

In preferred embodiments, described expression of nucleic acid feature comprises the nucleic acid molecule of at least a coding microrna sequences, and it is expressed in one or more target blood plasma to compare with one or more healthy individual, colorectal cancer and lung cancer and is raised; And the nucleic acid molecule that comprises at least a coding microrna sequences, it is expressed in one or more target blood plasma to compare with one or more healthy individual, colorectal cancer and lung cancer and is reduced.

In preferred embodiments, the expression of nucleic acid feature comprises coding: Tumor-assaciated feature hsa-miR-122, hsa-miR-192, hsa-miR-215, hsa-let-7c, hsa-miR-103, hsa-miR-139-3p, hsa-miR-26b; Plasma specific feature hsa-miR-936, hsa-miR-193b*, hsa-miR-124, hsa-miR-34a, hsa-miR-198, hsa-let-7g, hsa-miR-363 and internal stability contrast: any one of has-miR-1228 and hsa-miR-1238 or multiple nucleic acids molecule.

Particularly preferably, compare with healthy individual, colorectal cancer and lung cancer, any one or the multiple nucleic acids developed by molecule of hsa-miR-122, hsa-miR-192, hsa-miR-215, hsa-let-7c, hsa-miR-103, hsa-miR-26b, hsa-miR-34a, hsa-let-7g, hsa-miR-363 of encoding in one or more target blood plasma raised, and any one or the multiple nucleic acids developed by molecule of coding hsa-miR-936, hsa-miR-193b*, hsa-miR-124, hsa-miR-139-3p, hsa-miR-198 are reduced; Hsa-miR-1238 and hsa-miR-1228 do not change.

In a more preferred embodiment, the expression of nucleic acid feature comprises any one or the multiple nucleic acids molecule of codes for tumor correlated characteristic hsa-miR-122, hsa-miR-192, hsa-miR-215 and plasma specific feature hsa-miR-936, hsa-miR-193b* and hsa-miR-124.

Particularly preferably, compare with healthy individual, colorectal cancer and lung cancer, any one of encode in one or more target blood plasma hsa-miR-122, hsa-miR-192 and hsa-miR-215 or multiple nucleic acids developed by molecule are raised; And any one or the multiple nucleic acids developed by molecule of coding hsa-miR-936, hsa-miR-193b* and hsa-miR-124 are reduced.

In particularly preferred scheme, the expression of nucleic acid feature comprises coding hsa-miR-122/hsa-miR-936, hsa-miR-34a/hsa-miR-193b*, hsa-miR-34a/hsa-miR-198, hsa-miR-192/hsa-miR-936, hsa-miR-122/hsa-miR-193b*, hsa-miR-122/hsa-miR-198, hsa-miR-192/hsa-miR-124, hsa-miR-192/hsa-miR-193b*, hsa-miR-122/hsa-miR-124, hsa-miR-192/hsa-miR-198, hsa-miR-363/hsa-miR-936, hsa-miR-215/hsa-miR-193b*, hsa-miR-103/hsa-miR-936, hsa-miR-122/hsa-miR-139-3phsa-let-7c/hsa-miR-936, hsa-miR-215/hsa-miR-198, hsa-miR-192/hsa-miR-139-3p, hsa-miR-27b/hsa-miR-198, hsa-miR-26b/hsa-miR-936, hsa-let-7g/hsa-miR-936, hsa-miR-103/hsa-miR-198, hsa-let-7c/hsa-miR-193b*, hsa-miR-103/hsa-miR-193b*, hsa-miR-26b/hsa-miR-139-3p, hsa-let-7c/hsa-miR-198, hsa-miR-27b/hsa-miR-193b*, hsa-let-7g/hsa-miR-193b*, hsa-miR-363/hsa-miR-139-3p, hsa-let-7g/hsa-miR-198, hsa-miR-363/hsa-miR-198, hsa-miR-26b/hsa-miR-198, hsa-miR-103/hsa-miR-139-3p, hsa-miR-301a/hsa-miR-198, hsa-miR-26b/hsa-miR-193b*, hsa-let-7g/hsa-miR-139-3p, hsa-miR-363/hsa-miR-124, hsa-let-7c/hsa-miR-139-3p, hsa-miR-301a/hsa-miR-193b*, hsa-miR-301a/hsa-miR-139-3p, hsa-miR-26b/hsa-miR-124, hsa-let-7d/hsa-miR-198, hsa-let-7d/hsa-miR-139-3p, hsa-miR-103/hsa-miR-124, any one of hsa-miR-363/hsa-miR-193b* and hsa-let-7d/hsa-miR-193b* or multiple nucleic acids combination.

The third aspect, the present invention relates to for differentiating that one or more shows the method for the blood plasma of hepatocellular carcinoma, described method comprises: (a) determine the expression level of multiple nucleic acids molecule in described one or more target blood plasma, every kind of nucleic acid molecule encoding microrna sequences; (b) expression level of definite described multiple nucleic acids molecule in one or more normal healthy controls blood plasma; (c) expression level separately by contrasting in step (a) and obtaining (b), from described multiple nucleic acids molecule, identify one or more nucleic acid molecule of differential expression in described target blood plasma and contrast blood plasma, one or more nucleic acid molecule of wherein said differential expression represents feature defined herein (signature) together, the existence of its indication hepatocellular carcinoma.

In a preferred embodiment of the invention, described method comprises expression level of (a) definite kernel acid molecule combination in one or more target blood plasma, every kind of nucleic acid molecule microrna sequences of all encoding, and with specific formula calculating; (b) in one or more normal healthy controls blood plasma, determine the expression level that described nucleic acid molecule makes up, and calculate with specific formula; And (c) by relatively differentiating the described difference that is combined in described one or more target blood plasma at (a) and the expression level separately that (b) obtains in the step, all representative features of the combination of one or more differential expression wherein, the existence of its indication hepatocellular carcinoma.

In a more preferred embodiment, present method is further used for hepatocellular carcinoma and healthy individual, colorectal cancer and lung cancer are differentiated.

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

The 5th aspect the present invention relates to the method for prevention or treatment hepatocellular carcinoma, and described method comprises: the method that (a) limits with this paper is differentiated the expression of nucleic acid feature in blood plasma; The expression of one or more nucleic acid molecule of the coding microrna sequences that (b) the described expression of nucleic acid feature of change comprises in blood, described change is carried out in such a way, be that its expression of expressing the nucleic acid molecule that is raised in blood is reduced, and its expression of expressing the nucleic acid molecule of being reduced in blood is raised.

The 6th aspect, the present invention relates to the pharmaceutical composition for the hepatocellular carcinoma that prevents and/or treats blood, described composition comprises one or more nucleic acid molecule, every kind of equal encoding sequence of nucleic acid molecule, the at least part of complementation of microrna sequences that described sequence is coded with the nucleic acid molecule that its expression is raised in from the blood plasma of patients with hepatocellular carcinoma as herein defined, and/or described sequence is corresponding to it expresses the coded microrna sequences of nucleic acid molecule of being reduced in from the blood plasma of patients with hepatocellular carcinoma as herein defined.

At last, the 7th aspect the present invention relates to described pharmaceutical composition for the preparation of the purposes in the medicine that prevents and/or treats hepatocellular carcinoma.

It is clear that other embodiment of the present invention will become from the following detailed description.

The accompanying drawing summary

Fig. 1: schema is shown, and it has systematically illustrated the basic method steps of determining expression characteristic with reference to method of the present invention, is used for differentiating that one or more shows the target blood plasma of hepatocellular carcinoma.

Fig. 2: the human miRNA that is included in the particularly preferred expression characteristic of the present invention is shown, and described expression characteristic is respectively applied to differentiate one or more target blood plasma that represents hepatocellular carcinoma.Also illustrate with normal healthy controls among the figure and compare, the expression level of these miRNA and accuracy in hepatocellular carcinoma blood plasma (namely raising or downward modulation).Be used as the diagnostic biomarker in the combination of the first five six kinds of different miRNA and differentiate that accuracy in hepatocellular carcinoma and the normal healthy controls is at 87%-94%

Fig. 3: show the ROC tracing analysis (0: the normal healthy controls group of two the Tumor-assaciated miRNA (hsa-miR-122 and has-miR-139-3p) in a routine hepatocellular carcinoma and the normal healthy controls blood plasma; 1: the hepatocellular carcinoma group).The result shows that these miRNA are as susceptibility and the specificity of diagnostic biomarker.These contrast the miR-1238 standardization through the data that microarray (biochip) analysis obtains through internal stability.

Fig. 4: be illustrated in the human miRNA that is included on the other hand in the particularly preferred expression characteristic of the present invention, differentiate hepatocellular carcinoma and healthy individual, colorectal cancer and lung cancer with reference at a specified future date application the of the present invention.Also expression level and the accuracy (namely raising or downward modulation) of the miRNA in the patients with hepatocellular carcinoma compared in demonstration with healthy individual, colorectal cancer and lung cancer.Combination at the first five miRNA is used as the accuracy of diagnostic biomarker discriminating hepatocellular carcinoma at 85%-88%

Fig. 5: show by five kinds of different miRNA form four in the combination Comparison basis.The data variation that is obtained by microarray be 0.76 by quantitative dependency (R) between the data of real-time quantitative PCR (quantitative RT-PCR) acquisition.These results show that the miRNA feature that is obtained by Agilent miRNA microarray (Agilent miRNA microarray) is highly believable.

Detailed Description Of The Invention

The present invention is based on following unexpected discovery, be that hepatocellular carcinoma (HCC) can identify with high accuracy and susceptibility that by specific miRNA expression characteristic in the blood plasma wherein said expression characteristic typically comprises the human miRNA that is reduced by upper mediation as defined herein reliably.More particularly, described miRNA expression characteristic-by whole miRNA expression pattern in the analysed for plasma and/or each miRNA expression level-so that can detect HCC and discriminating healthy individual, colorectal cancer and lung cancer under the early stage morbid state.

The present invention of following illustration can be suitably implement under the condition of concrete any one or more element that discloses, one or more restriction in this article not existing.

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

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

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

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

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

Term further be defined in following use term the time provide.

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

An object of the present invention is to provide for diagnosing hepatocellular carcinoma, the treatment of monitoring cancer, and/or the novel method for the treatment of cancer, wherein by measuring the multiple nucleic acids molecule in the blood, every kind of nucleic acid molecule microRNA (miRNA) sequence of all encoding, one or more of wherein said multiple nucleic acids molecule in the blood plasma of hepatocellular carcinoma, compare with normal healthy controls by analysis and/or and healthy individual, colorectal cancer is compared differential expression with lung cancer, the nucleic acid molecule of wherein said one or more differential expression represents the expression of nucleic acid feature together, the existence of this expression of nucleic acid feature indication hepatocellular carcinoma, wherein said expression of nucleic acid feature comprises Tumor-assaciated feature and plasma specific feature.

Term used herein " cancer " (being also referred to as " cancer (carcinoma) ") is often referred to the malignant growth of any type, namely compares any morphology and/or the physiology change (based on hereditary reprogrammed (genetic re-programming)) that show or have the target cell that cancer feature tendency occurs with unaffected (health) wild-type control cells.The example of this change can relate to cell size and shape (become large or diminish), cell proliferation (cell count increase), cytodifferentiation (physiological status variation), apoptosis (apoptosis) or cell survival.

Term used herein " liver cell " relates to liver.Therefore, term " hepatocellular carcinoma " refers in hepatocellular cancerous growths.

The hepatocellular carcinoma of common type is hepatocellular carcinoma (is also referred to as liver cancer, usually is abbreviated as HCC).Term used herein " hepatocellular carcinoma " refers to former liver malignancy.The viral hepatitis that is secondary to most of HCC infects (hepatitis B or hepatitis C) or liver cirrhosis (alcoholism is the common factors that causes liver cirrhosis).In hepatitis was not in the right place the country of disease, most of liver malignant cancer were not former HCC, but from other position of body the cancer metastasis of colon (propagation) for example.Treatment selection and the prognosis of HCC depend on many factors, but depend on especially tumor size and by stages.Tumour grade (tumor grade) also is important.High-grade (high-grade) tumor prognosis is not good, and inferior grade (low-grade) tumour was also permitted a lot of years out in the cold.Normally result is not good to be because only the hepatocellular carcinoma of 10%-20% can be excised fully by operation.If cancer can not be excised fully, then this disease was fatal in 3-6 month usually.

Hepatocellular carcinoma is the same with any other cancer, when existence causes the cell high speed duplicating and/or causes cell to avoid molecular mechanism when sudden change of apoptosis and occur.Especially, the chronic viral infection of hepatitis B and/or hepatitis C is by repeating to cause body autoimmunization system attack liver cell (some of them are by virus infection, other only be the onlooker) and can help the generation of hepatocellular carcinoma.Although the mistake during the constant circulation of repairing after this damage can cause repairing causes oncogenesis thereupon, present this hypothesis more is applicable to hepatitis C.Yet in hepatitis B, it is the most uniformly correlated factor in the malignant tumour that viral genome is integrated in the cell of infection.In addition, repeat heavy drinking and can have similar action.

Therefore, in the scope of the invention, hepatitis B and/or hepatitis C infection or liver cirrhosis not only are considered to the Hazard Factor of tumor aetiology, but also be tumor development early stage/intergrade (i.e. " precancerous condition "), its with cause the excrescent excess proliferative tissue growth of (being generally optimum) Non-Invasive (thereupon can develop into malignant tumour such as HCC) relevant.

This malignant tumour is attacked other tissue and is often shifted when enough in the time.Malignant cell is characterised in that progressivity (progressive) and uncontrolled growth usually.Visual inspection HCC looks like nodositas or wettability tumour.The nodular type tumour can be single-shot (having agglomerate) or multiple (when developing into the sclerosis complication).Tumor nodule is circular to oval, clear border but without coating.The diffuse type obscure boundary, and invade the profit portal vein, seldom invade the profit hepatic vein.

The Mammals target blood plasma that adopts among the present invention can be people or non-human source.Yet the present invention typically carries out with human plasma.Term used herein " one or more blood plasma " should be understood to not only comprise individual blood plasma.Term used herein " target blood plasma " refers to that by assert at least be the blood plasma that shows or have the Risk of Hepatocellular Carcinoma tendency, and term " contrast blood plasma " typically refers to not have (health) type of this cancer phenotypic characteristic.For example when relatively showing the blood plasma of different carcinoma or precancerous condition, the cell with not too serious genius morbi typically is considered to " contrast blood plasma " but in some applications.

Term used herein " blood plasma " is the yellow liquid component in the blood, and the hemocyte in the whole blood can normally suspend therein.Its volume accounts for 55% of volume of whole blood.Most of composition is water (volume accounts for 90%) in the blood plasma, comprises soluble proteins, glucose, thrombin, mineral ion, hormone and carbonic acid gas (blood plasma is the main medium of movement transportation).It is by fresh blood is centrifugal in whizzer that blood plasma is prepared, until hemocyte sinks to the pipe end.Then blood plasma is inclined to.The density of blood plasma is probably at 1025kg/m ³, or 1.025kg/L.Recent research finds that miRNA is stable in blood plasma.Term " plasma sample " refers to the blood plasma to be checked that obtains from individuality or normal healthy controls.

Term used herein " patient " refers to should to be considered to suffer from least the people of hepatocellular carcinoma; Term used herein " target blood plasma " refers to the blood plasma that obtains from the patient; Term " healthy individual " or " normal healthy controls " are refered in particular to the healthy individual with arbitrary cancer performance.And " contrast blood plasma " refers to the blood plasma that obtains from these healthy individual here.But in some were used, for example, when more different type of cancer, these people had other type of cancer, and these blood plasma that obtain from these individualities are also refered in particular to is " contrast ".

Typically, used target blood plasma and contrast blood plasma are derived from the biological sample of collecting from wait to be diagnosed the object that whether has hepatocellular carcinoma or Risk of Hepatocellular Carcinoma tendency.In addition, in order to prove conclusively the data of acquisition, " comparative sample " also can be collected from the object of suffering from given known morbid state.Biological sample can comprise body tissue and liquid, such as tissue, serum, hemocyte, phlegm and urine.In addition, if necessary, cell can be from the body tissue that obtains and liquid purifying, then as biological sample.According to the present invention, the expression level of nucleic acid marking of the present invention is determined in the biological sample that object is derived

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

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 the genomic gene seat, and it is from forming the transcript processing of partial rna precursor miRNA structure.Ripe miRNA normal length is 20,21,22,23,24 or 25 Nucleotide, and the Nucleotide of other number also can exist, for example 18,19,26 or 27 Nucleotide.

The miRNA encoding sequence has the potentiality with the pairing of flanking gene group sequence, ripe miRNA is placed within the non-RNA duplex that matches fully (this paper is also referred to as stem-ring or hairpin structure or pre-miRNA), and described duplex is as the intermediate that carries out miRNA processing from longer precursor transcript.This processing typically continuous action of two species specific endonucleases by being called Drosha and Dicer occurs.Drosha produces the miRNA precursor (this paper is also referred to as " pre-miRNA ") that typically is folded into hair clip or stem-ring structure from primary transcript (this paper is also referred to as " pri-miRNA ").From this miRNA precursor, by Dicer cutting miRNA duplex, its one arm at hair clip or stem-ring structure comprises ripe miRNA, comprises the sections (being commonly referred to miRNA*) of similar size at other one arm.Then miRNA is directed to its said target mrna bringing into 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 process from it part of the miRNA primary transcript of ripe miRNA.Typically, pre-miRNA is folded into stable hair clip (being duplex) or stem-ring structure.Hairpin structure typically length is 50-80 Nucleotide, preferred 60-70 Nucleotide (counting miRNA residue, with the residue of miRNA pairing, and any sections that interleaves, but get rid of the more sequence of far-end).

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

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

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

Therefore, the multiple nucleic acids molecule that is included in the coding miRNA sequence in the diagnostic kit of the present invention can comprise that one or more " has the phosphorothioate odn molecule " and/or one or more " antisense nucleic acid molecule ".Sometimes, diagnostic kit comprises that one or more " has the phosphorothioate odn molecule " (being miRNA sequence itself), described molecule has been considered to form all or at least inferior set of the miRNA (being molecule marker) of differential expression, the miRNA of described differential expression is the indication that has or occur the particular disorder tendency, and this paper is hepatocellular carcinoma.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 be suitable for detecting and/or quantitative given sample in probe molecule (being used for carrying out hybridization assays) and/or the Oligonucleolide primers (for example being used for reverse transcription or PCR uses) of one or more specific (complementation) miRNA sequence.

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

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

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

Term used herein " expression level " refers to the degree that specific miRNA sequence is transcribed from 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 not have (health) blood plasma of HCC phenotypic characteristic.But in some applications, for example when relatively showing the blood plasma of different cancers or precancerous condition, the blood plasma with more not serious genius morbi typically is considered to " contrast blood plasma ".

The determining of expression level typically followed the standard program (Sambrook that has set up well known in the art, J.et al. (1989) Molecular Cloning:A Laborary Manual.2nd Ed., Cold Spring Harbor Library Press, Cold Spring Harbor, NY; Ausubel, F.M.et al. (2001) Current Pro-cols in Molecular Biology.Wiley ﹠amp; Sons, Hoboken, NJ).Determine and to carry out at rna level, for example use the miRNA specific probe to carry out the Northern engram analysis, perhaps behind reverse transcription (and clone) RNA group, for example carry out at dna level by quantitative PCR or real time pcr.Any nucleic acid molecule of the above-mentioned microrna sequences of analysis of encoding " determined " to comprise in term used herein.But, because pri-miRNA and re-mRNA transformation period are short, typically only measure the concentration of ripe miRNA.

In specific embodiment, the standard value of the expression level that obtains in some independent measurements (for example two, three, five or ten measurements) of given sample and/or the some measurements in a multiple targets blood plasma or contrast blood plasma is used to analyze.Standard value can obtain with any method known in the art.For example, the scope of mean value ± 2SD (standard deviation) or mean value ± 3SD can be used as standard value.

Difference between the expression level of institute's erworbene Krankenheit and contrast blood plasma can be normalized to for example expression level of house-keeping gene of further contrast nucleic acid, and the expression level of house-keeping gene is known not according to the morbid state of cell and difference.House-keeping gene for example comprises beta-actin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein P1 etc.In preferred embodiments, contrast nucleic acid be known in sample different non-cancer and cancer (front) state in the another kind of miRNA of stably express.

But, replace in any experiment, determining the expression level of one or more contrast blood plasma, also can be based on experimental evidence and/or prior art data definition one or more cutoff value for specified disease phenotype (being morbid state).In this case, the corresponding expression level of one or more target blood plasma can be determined with the contrast miRNA that is used for normalized stably express.If the expression level of " normalization method " of calculating is higher than the cutoff value of corresponding definition, then this discovery is the indication that genetic expression is raised.Otherwise if the expression level of " normalization method " of calculating is lower than the cutoff value of corresponding definition, then this discovery is the indication of down regulation of gene expression.

In the present invention, term " is identified hepatocellular carcinoma and/or is differentiated other type of cancer " and also comprises prediction and probability analysis (on " diagnosis " meaning).Composition disclosed herein and method are intended to clinical application, to determine form of therapy, comprise therapeutic intervention, Case definition such as disease stage, and disease surveillance and surveillance of disease.According to the present invention, can be provided for checking the intermediate result of Obj State.This intermediate result can make up to help doctor, nurse or other practitioner to diagnose out this object to suffer from this disease with extraneous information.Perhaps, the present invention can be used for detecting by blood sample the change of cancer tendency, and provide useful information to the doctor to diagnose.In addition, the present invention also is used for distinguishing dissimilar liver cell tumor and other type of cancer comprise colorectal cancer and lung cancer.

In the present invention, the nucleic acid molecule of one or more differential expression of identifying represents a kind of expression of nucleic acid feature together, and this expression of nucleic acid feature is the indication that has hepatocellular carcinoma in target blood plasma.Term used herein " expression characteristic " refers to one group of nucleic acid molecule (for example miRNA), and wherein the expression level of each nucleic acid molecule is different between (carcinous) target blood plasma and (non-carcinous) contrast blood plasma.Herein, the expression of nucleic acid feature also refers to a group echo and represents minimum purpose (difference) nucleic acid molecule that every kind of nucleic acid molecule encoding can be identified the miRNA sequence of the phenotype state of target blood plasma.

In preferred embodiments, described expression of nucleic acid feature comprises the nucleic acid molecule of at least a coding microrna sequences, and it is expressed in one or more target blood plasma to compare with one or more contrast blood plasma and is raised; And the nucleic acid molecule that comprises at least a coding microrna sequences, its expression is compared with one or more contrast and is reduced

The term of this paper " plasma specific " (plasma-specific) refers to feature in the variant expression of blood plasma of liver cancer patient and normal control, but does not find significant differential expression between Tissues of Hepatocellular Carcinoma cell and non-cancer tissue cell.

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

In a preferred embodiment of the invention, the expression of nucleic acid feature comprises the hsa-miR-122 (SEQ ID NO:1) that codes for tumor is relevant, hsa-miR-199b-3p (SEQ ID NO:2), hsa-miR-192 (SEQ ID NO:3), hsa-miR-21 (SEQ ID NO:4), hsa-miR-139-3p (SEQ ID NO:5), hsa-miR-10a (SEQ ID NO:6), hsa-miR-103 (SEQ ID NO:7), hsa-miR-181d (SEQ ID NO:8), hsa-miR-125b-2* (SEQ ID NO:9), the expression of any one of hsa-miR-125a-3p (SEQ ID NO:10) or multiple nucleic acids molecule; The hsa-miR-34a (SEQ ID NO:11) of coding plasma specific, hsa-miR-136 (SEQ ID NO:12), hsa-miR-151-5p (SEQ ID NO:13), hsa-miR-193b* (SEQ ID NO:14), hsa-miR-124 (SEQ ID NO:15), hsa-miR-936 (SEQ ID NO:16), hsa-miR-198 (SEQ ID NO:17), hsa-miR-149* (SEQ ID NO:18), hsa-miR-138 (SEQ ID NO:19), hsa-miR-601 (SEQ ID NO:20), hsa-miR-769-3p (SEQ ID NO:21), hsa-miR-513c (SEQ ID NO:22), hsa-miR-525-5p (SEQ ID NO:23), hsa-miR-654-5p (SEQ ID NO:24), hsa-miR-518c* (SEQ ID NO:25), hsa-miR-500 (SEQ ID NO:26), hsa-miR-181c* (SEQ ID NO:27), hsa-miR-1226* (SEQ ID NO:28), hsa-miR-202 (SEQ ID NO:29), the expression of hsa-miR-629* (SEQ ID NO:30) any one or multiple nucleic acids molecule; Coding is stablized the hsa-miR-1238 (SEQ ID NO:36) of internal stability contrast and the nucleic acid molecule of hsa-miR-1228 (SEQ ID NO:37).

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

Table 1

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

Particularly preferably, with compare in blood plasma in one or more contrast, hsa-miR-122 encodes in described one or more target blood plasma, hsa-miR-199b-3p, hsa-miR-192, hsa-miR-21, hsa-miR-10a, hsa-miR-103, hsa-miR-34a, hsa-miR-136, the expression of any one of hsa-miR-151-5p or multiple nucleic acids molecule is raised, and coding hsa-miR-139-3p, hsa-miR-193b*, hsa-miR-124, hsa-miR-181d, hsa-miR-125b-2*, hsa-miR-125a-3p, hsa-miR-936, hsa-miR-198, hsa-miR-149*, hsa-miR-138, hsa-miR-601, hsa-miR-769-3p, hsa-miR-513c, hsa-miR-525-5p, hsa-miR-654-5p, hsa-miR-518c*, hsa-miR-500, hsa-miR-181c*, hsa-miR-1226*, hsa-miR-202, the expression of any one of hsa-miR-629* or multiple nucleic acids molecule is reduced; Hsa-miR-1238 and hsa-miR-1228 are unchanged.

As used herein, term " one or more of described multiple nucleic acids molecule " reaches any subgroup that " nucleic acid molecule that any one or various human target cell derive " can relate to described multiple nucleic acids molecule, such as any, 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, every kind of equal encoded packets of nucleic acid molecule is contained in the microrna sequences in the described expression of nucleic acid feature.

In particularly preferred embodiments, adjust expression characteristic and comprise coding hsa-miR-34a/hsa-miR-193b*, hsa-miR-21/hsa-miR-936, hsa-miR-192/hsa-miR-124, hsa-miR-122/hsa-miR-193b*, hsa-miR-34a/hsa-miR-138, hsa-miR-34a/hsa-miR-198, hsa-miR-122/hsa-miR-124, hsa-miR-103/hsa-miR-139-3p, hsa-miR-192/hsa-miR-193b*, hsa-miR-122/hsa-miR-138, hsa-miR-34a/hsa-miR-139-3p, hsa-miR-122/hsa-miR-198, hsa-miR-122/hsa-miR-769-3p, hsa-miR-103/hsa-miR-193b*, hsa-miR-34a/hsa-miR-124, hsa-miR-192/hsa-miR-139-3p, hsa-miR-34a/hsa-miR-769-3p, hsa-miR-192/hsa-miR-936, hsa-miR-10a/hsa-miR-193b*, hsa-miR-122/hsa-miR-601, hsa-miR-21/hsa-miR-769-3p, hsa-miR-199b-3p/hsa-miR-193b*, hsa-miR-103/hsa-miR-138, hsa-miR-103/hsa-miR-198, hsa-miR-199b-3p/hsa-miR-139-3p, the combination of any one of hsa-miR-21/hsa-miR-139-3p and hsa-miR-21/hsa-miR-193b* or multiple nucleic acids developed by molecule.

Term used herein " nucleic acid combination " refers to two or more expression of nucleic acid levels are done as a whole use.Particularly use dependency to change or with same one-piece pattern calculation result.

In preferred embodiments, the expression of nucleic acid feature comprises coding: Tumor-assaciated feature hsa-miR-122 (SEQ ID NO:1), hsa-miR-192 (SEQ ID NO:4), hsa-miR-215 (SEQ ID NO:31), hsa-let-7c (SEQ ID NO:32), hsa-miR-103 (SEQ ID NO:5), hsa-miR-139-3p (SEQ IDNO:7), hsa-miR-26b (SEQ ID NO:33); Plasma specific feature hsa-miR-936 (SEQID NO:16), hsa-miR-193b* (SEQ ID NO:14), hsa-miR-124 (SEQ ID NO:15), hsa-miR-34a (SEQ ID NO:11), hsa-miR-198 (SEQ ID NO:18), hsa-let-7g (SEQ ID NO:34), any one of hsa-30miR-363 (SEQ ID NO:35) and internal stability contrast has-miR-1228 (SEQ ID NO:36) and hsa-miR-1238 (SEQ ID NO:37) or multiple nucleic acids molecule.

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

Table 2

Particularly preferably, with compare in one or more healthy individual, colorectal cancer and lung cancer, any one of encode in described one or more target blood plasma hsa-miR-122, hsa-miR-192, hsa-miR-215, hsa-let-7c, hsa-miR-103, hsa-miR-26b, hsa-miR-34a, hsa-let-7g, hsa-miR-363 or multiple nucleic acids developed by molecule are raised; And any one or the multiple nucleic acids developed by molecule of coding hsa-miR-936, hsa-miR-193b*, hsa-miR-124, hsa-miR-139-3p and hsa-miR-198 are reduced; Hsa-miR-1238 and hsa-miR-1228 do not change.

In particularly preferred embodiments, the expression of nucleic acid feature comprises coding hsa-miR-122/hsa-miR-936, hsa-miR-34a/hsa-miR-193b*, hsa-miR-34a/hsa-miR-198, hsa-miR-192/hsa-miR-936, hsa-miR-122/hsa-miR-193b*, hsa-miR-122/hsa-miR-198, hsa-miR-192/hsa-miR-124, hsa-miR-192/hsa-miR-193b*, hsa-miR-122/hsa-miR-124, hsa-miR-192/hsa-miR-198, hsa-miR-363/hsa-miR-936, hsa-miR-215/hsa-miR-193b*, hsa-miR-103/hsa-miR-936, hsa-miR-122/hsa-miR-139-3p, hsa-let-7c/hsa-miR-936, hsa-miR-215/hsa-miR-198, hsa-miR-192/hsa-miR-139-3p, hsa-miR-27b/hsa-miR-198, hsa-miR-26b/hsa-miR-936, hsa-let-7g/hsa-miR-936, hsa-miR-103/hsa-miR-198, hsa-let-7c/hsa-miR-193b*, hsa-miR-103/hsa-miR-193b*, hsa-miR-26b/hsa-miR-139-3p, hsa-let-7c/hsa-miR-198, hsa-miR-27b/hsa-miR-193b*, hsa-let-7g/hsa-miR-193b*, hsa-miR-363/hsa-miR-139-3p, hsa-let-7g/hsa-miR-198, hsa-miR-363/hsa-miR-198, hsa-miR-26b/hsa-miR-198, hsa-miR-103/hsa-miR-139-3p, hsa-miR-301a/hsa-miR-198, hsa-miR-26b/hsa-miR-193b*, hsa-let-7g/hsa-miR-139-3p, hsa-miR-363/hsa-miR-124, hsa-let-7c/hsa-miR-139-3p, hsa-miR-301a/hsa-miR-193b*, hsa-miR-301a/hsa-miR-139-3p, hsa-miR-26b/hsa-miR-124, hsa-let-7d/hsa-miR-198, hsa-let-7d/hsa-miR-139-3, hsa-miR-103/hsa-miR-124, any one of hsa-miR-363/hsa-miR-193b* and hsa-let-7d/hsa-miR-193b* or multiple nucleic acids combination.

As used herein, term " changes the expression of the nucleic acid molecule of coding miRNA sequence " and refers to any manipulation of specific nucleic acid molecule is changed with the expression level that causes described molecule, namely compares the corresponding miRNA that produces different amounts from the expression of " wild-type " (being unaltered contrast).As used herein, term " different amount " had both comprised the high amount of comparing 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 downward modulation (namely suppressing) nucleic acid molecule.

In the present invention, the expression of one or more nucleic acid molecule of the coding microrna sequences that comprises in the expression of nucleic acid feature is modified by this way, and its expression that expression of expressing the nucleic acid molecule that is raised in described one or more target blood plasma is reduced and it expresses the nucleic acid molecule of being reduced in described one or more target blood plasma is raised thus.In other words, the modification of the expression of the specific nucleic acid molecule of coding miRNA sequence with the generation of the antimode (anti-cyclical) of the regulating effect of described molecule in described one or more cancer target blood plasma, with " overactivity " of in described one or more target blood plasma, disturbing the molecule that is raised and/or recover " defective is active " of the molecule reduced.

In a preferred embodiment of the inventive method, the expression of downward modulation nucleic acid molecule comprise will coding with imported in the patient body by the nucleic acid molecule of the sequence of the microrna sequences complementation of the nucleic acid molecule encoding reduced.

Term as used herein " imports in the blood " and refers to so that one or more nucleic acid molecule shifts any manipulation that enters in the blood.The example of this technology comprises injection well known in the art, digestion and other technologies.

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

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

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

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

Developed recently another kind of miRNA expression silencing gene.These chemical engineering oligonucleotide that are called " antagomirs " are RNA molecules (Krutzfeldt, J.et al. (2005) Nature 438,685 – 689) of 23 Nucleotide of strand of puting together with cholesterol.Select as another of this chemically modified oligonucleotide, produced as RNA from transgenosis, produce can be at the microRNA inhibitor of cells.These competitive inhibitors that are called " microRNA sponge (microRNA sponges) " are the transcripts of expressing from strong promoter, the a plurality of series combination site (Ebert that contains interested microRNA, M.S.et al. (2007) Nat.Methods 4,721-726).

In the particularly preferred embodiment of the inventive method, it is expressed with respect to expression characteristic and one or more nucleic acid molecule encoding of being reduced is selected from the microrna sequences such as next group: hsa-miR-139-3p, hsa-miR-181d, hsa-miR-125b-2*, hsa-miR-125a-3p, hsa-miR-193b*, hsa-miR-124, hsa-miR-936, hsa-miR-138, hsa-miR-198, hsa-miR-769-3p, hsa-miR-149*, hsa-miR-654-5p, hsa-miR-525-5p, hsa-miR-629*, hsa-miR-181c*, hsa-miR-202, hsa-miR-513c, hsa-miR-500, hsa-miR-518c*, hsa-miR-601 and hsa-miR-1226* may be the indication of hepatocellular carcinoma as mentioned above.

In another preferred embodiment of the inventive method, raise nucleic acid molecule and express in the nucleic acid molecule importing blood of the microrna sequences that comprises the nucleic acid molecule encoding that coding is raised.In other words, the rise of the expression of the nucleic acid molecule of coding miRNA sequence imports in described one or more cell by another copy (being other " justice is arranged " nucleic acid molecule) with described miRNA sequence and realizes.Described " justice is arranged " nucleic acid molecule that imports 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 with respect to expression characteristic and one or more nucleic acid molecule encoding of being raised is selected from the microrna sequences such as next group: hsa-miR-122, hsa-miR-199b-3p, hsa-miR-192, hsa-miR-21, hsa-miR-10a, hsa-miR-103, hsa-miR-34a, hsa-miR-136, hsa-miR-151-5p, hsa-miR-215, hsa-let-7c, hsa-miR-26b, hsa-let-7g and hsa-miR-363 may be the indication of hepatocellular carcinoma as mentioned above.

Import in one or more target cell with " justice is arranged " and/or " antisense " nucleic acid molecule of the expression of the nucleic acid molecule of modifying the microrna sequences that is comprised in one or more coding nucleic acid expression characteristic can with regulate that sequence operably be connected so that described nucleotide sequence is expressed.

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

If nucleic acid molecule comprises the sequential element that contains relevant for transcribing and/or translate adjusting information, and this sequence " operably connects " in the nucleotide sequence of coded polypeptide, then claims this nucleic acid molecule to be " energy express nucleic acid molecule " or energy " so that nucleotide sequence expression ".Operably connect be wherein said adjusting sequential element with the sequence that is expressed (and/or sequence of expressing mutually) with energy so that the connection that the mode of genetic expression is connected.

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

In addition, as defined herein the expression of nucleic acid molecule also can affect (as mentioned above) by the Nucleotide of for example exist modifying.For example, locked nucleic acid (LNA) monomer is considered to increase in the body the functional transformation period of miRNA for reticent active crucial miRNA-target duplex structure and (see for example Naguibneva by strengthening to the resistance of degraded and by stable, I.et al. (2006) Biomed.Pharmacother.60,633 – 638).

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

Nucleic acid molecule of the present invention also can be included in carrier or other cloning vector such as plasmid, phagemid, phage, clay or the artificial chromosome.In preferred embodiments, described nucleic acid molecule is included in the carrier, particularly is included in the expression vector.This expression vector except the nucleotide sequence of the genetic constructs of above-mentioned adjusting sequence and coding as the present invention definition, can comprise derived from the selective marker that can select phenotype with control sequence and the cell of giving transfection that copies for the compatible species of the host of expression.Many suitable carriers known in the art and commercially available such as pSUPER and pSUPERIOR.

The 6th aspect, the present invention relates to the pharmaceutical composition for the hepatocellular carcinoma that prevents and/or treats blood, described composition comprises one or more nucleic acid molecule, every kind of equal encoding sequence of nucleic acid molecule, the at least part of complementation of microrna sequences that described sequence is coded with the nucleic acid molecule that its expression is raised in from the blood plasma of patients with hepatocellular carcinoma as herein defined, and/or described sequence is corresponding to it expresses the coded microrna sequences of nucleic acid molecule of being reduced in from the blood plasma of colorectal cancer patients as herein defined.

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

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

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

For pharmaceutical compositions, can use the inorganic or organic excipients (being carrier) of pharmacy inertia.For preparation example such as pill, tablet, capsule or particle, can use for example lactose, talcum, stearic acid and salt thereof, fat, wax, solid or liquid polyol, natural oil or winterized stearin.For the production of solution, suspension, milk sap, aerosol mixture or before using reprovision comprise water, alcohol, glycerine, polyvalent alcohol and suitable mixture and vegetables oil thereof as the appropriate excipients of the powder of solution or aerosol mixture.

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

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

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

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

The present invention further describes by accompanying drawing and following embodiment, and described drawings and Examples just for the purpose of illustration special embodiment of the present invention, should not be construed as the meaning that limits by any way the scope of the invention.

Embodiment

Embodiment 1: sample collection and preparation

63 routine hepatic tissue excision samples.Immediately quick-frozen is in liquid nitrogen after collecting quick-frozen or collection in the surgical operation sample liquid nitrogen.Sample is stored at-80 ℃.The essential characteristic of selected tumor sample is as shown in table 3.

The essential characteristic of table 3 hepatic tissue sample

Tissue sample	Number
		Normal liver tissue	31
The HCC tissue	32
		Sum	63

Patient's data (age, sex, image data, treatment and other medical condition, family history suchlike) obtains to mate collected different samples in the database of hospital.The pathology follow is (for example by h and E (H﹠amp; E) histologic analysis that dyes and carry out) be used for clearly determining the morbid state (i.e. contrast, precancer (for example liver cirrhosis), primary malignant neoplasm (for example hepatocellular carcinoma) or secondary/transfering malignant tumor (for example cancer embolus)) of given sample and the consistent classification that guarantees sample.

Carry out the laser capture micro-dissections with specific isolation tumor cell group (about 200000 cells) to each cancer sample is optional.In brief, transparent transfer film is applied to the surface of tissue slice or sample.At microscopically, observe the thin tissue section that places on the slide glass, and the identification of cell group is to separate.When the cell of selecting is positioned at the center of field of view, activate the integrated Optics in Microscope device of near-infrared laser diode (near IR laser diode integral with the microscope optics).Pulse laser beam activates the spot (spot) on the transfer film, makes the cytogamy of this film and following selection.The transfer film that then will have the cell of combination is peeled off (Emmert-Buck, M.R.et al. (1996) .Science 274,998-1001 from described thin tissue section; Espina, V.et al. (2007) Expert Rev.Mol.Diagn.7,647-657).Basic as manufacturer's guidance system is for freezing microtome section and use laser capture microscope (Arcturus Veritas ^TMLaser Capture Microdissection Instrument (Molecular Devices, Inc., Sunnyvale, CA, USA) catches step.

Use manufacturer (Ambion, Austin, TX). mirVana miRNA extraction agent box (mirVana miRNA isolation kit) obtain total RNA.Measure total RNAA concentration with NanoDrop 1000 spectrophotometers (NanoDrop Technologies, Waltham, MA).Quality monitoring to RNA then uses RNA 6000 Pico LabChip test kits (Agilent Technologies, Santa Clara, CA) to realize by 2100 Bioanalyzer.

Embodiment 2; The analysis that miRNA expresses in the tissue samples

Use Agilent miRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA) to instruct the optional miRNA that (difference) in the specific sample is expressed to carry out qualitative analysis according to manufacturer.By using the Quantile method and using R software known in the art to hybridize the raw data normalization method that obtains for monochromatic (CY3).

The well-known Quantile method in this field and GeneSpring GX10 software (Agilent Technologies, Santa Clara, CA, USA) normalization method are used by the raw data that monochromatic (CY3) obtains in the data analysis aspect.Fisher check (F-test) non-matching t-afterwards checks (p＜0.01), is used for determining the differential expression of miRNA between normal liver tissue and the HCC tissue.

63 routine tissue samples all are independently to test to detect, and the expression level of miRNA is decided by to obtain separately the mean value of data.

Embodiment 3: the collection of plasma sample and preparation

Fig. 1 is shown in the committed step of the indication that defines hepatocellular carcinoma in the blood samples of patients.

We collect blood sample 154 examples of 2008-2009 cancer and healthy individual in upper marine mountain hospital and Huashan hospital.The essential characteristic of the blood sample that the present invention is used as shown in Figure 4.All patients' blood sample all obtains before operation.Patient data (age, sex, image data, treatment and other medical condition, family history suchlike) obtains in the database of hospital.The histopathologic classification of tumour is distinguished complete independentlies with reference to staging system of the World Health Organization (WHO) by three Pathology Doctors 's.

The essential characteristic of table 4 blood preparation

The miRNA of genome range analyzes	Number
		Contrast	?
Healthy individual	23
		Colorectal cancer	31
Lung cancer	36
		Hepatocellular carcinoma	24
?	?
		Quantitative RT-PCR is analyzed	?
Healthy individual	16
		Hepatocellular carcinoma	24
Sum	154

Take peripheral blood (2ml) to containing in the pipe that EDTA contains, want centrifugal 820 to turn 10 minutes in two hours.Then the centrifuge tube centrifugal 16000 that blood plasma that will about 1ml is transferred to 1.5ml turns 10 minutes to precipitate any remaining cell debris.Then supernatant liquor is moved to-80 ℃ of preservations in the new pipe.

Use mirVana PARIS miRNA extraction agent box (mirVana PARIS miRNA isolation kit) to obtain total RNA with reference to the operation instruction of manufacturer (Ambion, Austin, TX).Measure total rna concentration with NanoDrop 1000 spectrophotometers (NanoDrop Technologies, Waltham, MA).Quality monitoring to RNA then uses RNA 6000 Pico LabChip test kits (Agilent Technologies, Santa Clara, CA) to realize by 2100 Bioanalyzer.

Embodiment 4: miRNA expression pattern analysis in the blood plasma

Use Agilent miRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA) to instruct the optional miRNA that (difference) in the specific blood product is expressed to carry out qualitative analysis according to manufacturer.This microarray is taken from v.10.1 database of Sanger, comprises 723 human miRNA probes.In 114 blood total RNA (100ng) of each sample extracting with the Cy3 mark after loading.And will cross XDRScan (PMT100, PMT5) scanning and obtain signal.The method of mark and hybridization is all with reference to Agilent miRNA microarray specification sheets.

Internal reference (hsa-miR-1238) normalization method is stablized by the raw data application that monochromatic (CY3) obtains in the data analysis aspect.Fisher check (F-test) non-matching t-check (p＜0.01) afterwards, be used for determining between normal liver tissue and the HCC tissue and or HCC with respect to the differential expression of healthy individual, colorectal cancer and lung cancer miRNA.

Use MedCalc software to HCC relatively with normal healthy controls and or HCC carry out recipient's performance characteristic with respect to single miRNA in healthy individual, colorectal cancer and the lung cancer (susceptibility and the specificity analyses of single miRNA as diagnosing biomarker analyzed in (ROC) tracing analysis.Limiting significant credibility interval is 95%.

What follows standard whether evaluate some miRNA HCC relatively with normal healthy controls with or HCC with respect to healthy individual, colorectal cancer and lung cancer in variant expression.

(i) the p value (probable value)≤0.05 of differential expression 〉=2 times change; And

(ii) AUC (as the accuracy of diagnostic biomarker)〉0.700

If these standards satisfy at least, then think described miRNA HCC relatively with normal healthy controls with or HCC with respect to healthy individual, colorectal cancer and lung cancer in variant expression.

114 routine blood all are independently to test to detect, and the expression level of miRNA is decided by to obtain separately the mean value of data.

HCC is with respect to normal healthy controls miRNA differential expression analytical data shown in the below's table 5-7.Table 5 is listed in HCC and the normal healthy controls miRNA differential expression analysis in tissue and the blood plasma.Table 6 has been summed up miRNA differential expression analysis in the blood plasma in HCC and the normal healthy controls.Table 7 has been listed characteristic miRNA combination best in HCC patient's blood plasma.The first five combined diagnosis biomarker that is formed by six different miRNA differentiate hepatocellular carcinoma and health accuracy at 87%-94%.In the table hurdle, " t " represents the HCC tissue, and " n " represents normal liver tissue, and " p " represents HCC patient, and " h " represents normal healthy controls.Particularly preferred miRNA (the SEQ ID NO:1-7 in the table 5 and the SEQ ID NO:11-17 in the table 6) is also marked with boldface type.

Tumor-assaciated miRNA feature in the blood plasma of table 5 hepatocellular carcinoma

The plasma specific miRNA feature of table 6 hepatocellular carcinoma

The miRNA feature of the combination in the blood plasma of table 7 hepatocellular carcinoma

Show among second aspect such as the following table 8-10 and differentiate hepatocellular carcinoma and healthy individual, colorectal cancer and the preferred specific expressed data of lung cancer.Table 8 is listed the Tumor-assaciated miRNA expression characteristic of hepatocellular carcinoma; Table 9 shows plasma specific miRNA expression characteristic; And table 10 has been listed characteristic miRNA combination best in HCC patient's blood plasma.The first five differentiates that by the combined diagnosis biomarker that six different miRNA form the accuracy of hepatocellular carcinoma and healthy individual, colorectal cancer and lung cancer is at 85%-88%.In the table hurdle, " t " represents the HCC tissue, and " n " represents normal liver tissue, and the blood plasma that " HCC " representative obtains from HCC patient, the blood plasma that " contrast " representative is obtained from healthy individual.Particularly preferred miRNA (the SEQ ID NO:1 in the table 8, SEQ ID NO:4, SEQ ID NO:31; SEQ ID NO:14-SEQ ID NO:16 in the table 9) also marked with boldface type.

Table 8 is used for the Tumor-assaciated miRNA expression characteristic with hepatocellular carcinoma and healthy individual, colorectal cancer and lung cancer difference

Table 9 is used for the plasma specific miRNA expression characteristic with hepatocellular carcinoma and healthy individual, colorectal cancer and lung cancer difference

Table 10 is used for the combination miRNA expression characteristic with hepatocellular carcinoma and healthy individual, colorectal cancer and lung cancer difference

Embodiment 5: the checking of microarray data

For verify (with or quantitatively) data expressed of the miRNA that obtains of microarray, adopt TaqMan MicroRNA to analyze (Applied Biosystems, Foster City, CA, USA) the real-time quantitative RT-PCR technology is with reference to its specification sheets (operation).Briefly, select the Taqman microRNA RT Kits of Applied Biosystem to carry out reverse transcription (RT) with reference to its explanation number.Comprise total RNA 10ng, 1X reverse transcription damping fluid, 1X RT primer, 1nM dNTP, 4U RNA enzyme inhibitors and 50U MultiScribe reversed transcriptive enzyme in the 15ul RT system.Then the RT system solution is carried out following program at PCR instrument (Thermal cycler alpha engine, Bio-rad): 16 ° of C, 30min; 42 ° of C, 30min; 85 ° of C, 5min.Quantitative PCR is then selected TaqMan Universal PCR Master Mix test kit and Taqman microRNA to measure the test kit such as test kit and is used with reference to its explanation.The quantitative PCR system comprises RTT product, 1X TaqMan Universal PCR Master Mix (No AmpErase) UNG, the 1X TaqMan MicroRNA Assay mix of 2ul.Each reacts triplicate.Following program is carried out and carried out to PCR in real time at Roch Light Cycling 480: 96 ° of C of elder generation, 5min; Then 45 or 50 circulations, 95 ° of C, 15s; 60 ° of C, 60s.The Cp value is finished at LC480 software with the method for second derivative (2nd derivative).The Cp value of each miRNA is with stablizing confidential reference items hsa-miR-1228 normalization method.

Be shown the experimental data of the contrast platform of 4 miRNA combinations that 5 different miRNA in the blood plasma that derives from 16 normal healthy controls and HCC patient form such as Fig. 4.The dependency of the data multiple varied number of obtaining by microarray with by PCR in real time is 0.76.The expression characteristic that the miRNA that Agilent miRNA microarray obtains is described is highly believable.

These results show that the expression level of miRNA liver cancer patient has the specificity of height in the whole world.Dimension each specificity miRNA is herein representing unique miRNA expression characteristic, diagnosing hepatocellular carcinoma, not only can diagnosing liver cancer tendency state and also can differentiate it and colorectal cancer and lung cancer.

The miRNA characteristic of identifying among the present invention be expressed as with blood test screen, discovery and diagnosing hepatocellular carcinoma provide distinctive molecule marker.And these characteristics expression also are used to monitor liver cancer patient therapeutic response and guiding treatment scheme.In addition, these characteristics are expressed and can also be used to develop medicines resistant to liver cancer.

This describe for example the present invention can suitably not exist under the condition of the special any element that discloses of this paper, restriction and carrying out.Therefore, such as term " comprise ", " comprising " " contain " etc. and should have broad sense and unrestricted.In addition; the term that this paper uses and expression are used for describing the present invention and unrestricted meaning; and do not use these terms and express to get rid of the meaning of any feature shown in it and description or its a part of Equivalent, but should recognize in the scope of the invention of asking for protection and to carry out various modifications.Therefore, although should understand the special announcement of the present invention being carried out by embodiment and optional feature, those skilled in the art can make amendment and change the present invention, and this modification and change are thought within the scope of the invention.

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

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

Claims

1. for the diagnostic kit of the blood molecular marked compound of differentiating the target blood plasma that one or more shows hepatocellular carcinoma, described test kit comprises the multiple nucleic acids molecule, every kind of nucleic acid molecule encoding microrna sequences,

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

The nucleic acid molecule of wherein said one or more differential expression represents the expression of nucleic acid feature together, the existence of described expression of nucleic acid feature indication hepatocellular carcinoma.

2. the test kit of claim 1, wherein said expression of nucleic acid feature can comprise at least three ten two kinds of nucleic acid molecule, preferably at least six kinds of nucleic acid molecule.

3. claim 1 or 2 test kit, wherein said expression of nucleic acid feature comprises the nucleic acid molecule of at least a coding microrna sequences, and it is expressed in one or more target blood plasma with comparing in one or more contrast blood plasma and is raised; And comprising the nucleic acid molecule of at least a coding microrna sequences, it is expressed in one or more target blood plasma with comparing in one or more contrast blood plasma and is reduced.

4. each test kit of claim 1-3, wherein said expression of nucleic acid feature comprises codes for tumor correlated characteristic hsa-miR-122, hsa-miR-199b-3p, hsa-miR-192, hsa-miR-21, hsa-miR-139-3p, hsa-miR-10a, hsa-miR-103, hsa-miR-181d, hsa-miR-125b-2*, a-miR-125a-3p; Plasma specific feature hsa-miR-34a, hsa-miR-136, hsa-miR-151-5p, hsa-miR-193b*, hsa-miR-124, hsa-miR-936, hsa-miR-198, hsa-miR-149*, hsa-miR-138, hsa-miR-601, hsa-miR-769-3p, hsa-miR-513c, hsa-miR-525-5p, hsa-miR-654-5p, hsa-miR-518c*, hsa-miR-500, hsa-miR-181c*, hsa-miR-1226*, hsa-miR-202, any one of the contrast hsa-miR-1238 of hsa-miR-629* and internal stability and hsa-miR-1228 or multiple nucleic acids molecule.

5. the test kit of claim 4, wherein compare with one or more normal healthy controls, in described one or more target blood plasma: the expression of any one of coding hsa-miR-122, hsa-miR-199b-3p, hsa-miR-192, hsa-miR-21, hsa-miR-10a, hsa-miR-103, hsa-miR-34a, hsa-miR-136, hsa-miR-151-5p or multiple nucleic acids molecule is raised; Coding hsa-miR-139-3p, hsa-miR-193b*, hsa-miR-124, hsa-miR-181d, hsa-miR-125b-2*, hsa-miR-125a-3p, hsa-miR-936, hsa-miR-198, hsa-miR-149*, hsa-miR-138, hsa-miR-601, hsa-miR-769-3p, hsa-miR-513c, hsa-miR-525-5p, hsa-miR-654-5p, hsa-miR-518c*, hsa-miR-500, hsa-miR-181c*, hsa-miR-1226*, hsa-miR-202, any one of hsa-miR-629* or multiple nucleic acids developed by molecule are reduced; Hsa-miR-1238 and hsa-miR-1228 do not change.

6. sharp each the test kit of 1-3 that requires, wherein said expression of nucleic acid feature comprises coding hsa-miR-34a/hsa-miR-193b*, hsa-miR-21/hsa-miR-936, hsa-miR-192/hsa-miR-124, hsa-miR-122/hsa-miR-193b*, hsa-miR-34a/hsa-miR-138, hsa-miR-34a/hsa-miR-198, hsa-miR-122/hsa-miR-124, hsa-miR-103/hsa-miR-139-3p, hsa-miR-192/hsa-miR-193b*, hsa-miR-122/hsa-miR-138, hsa-miR-34a/hsa-miR-139-3p, hsa-miR-122/hsa-miR-198, hsa-miR-122/hsa-miR-769-3p, hsa-miR-103/hsa-miR-193b*, hsa-miR-34a/hsa-miR-124, hsa-miR-192/hsa-miR-139-3p, hsa-miR-34a/hsa-miR-769-3p, hsa-miR-192/hsa-miR-936, hsa-miR-10a/hsa-miR-193b*, hsa-miR-122/hsa-miR-601, hsa-miR-21/hsa-miR-769-3p, hsa-miR-199b-3p/hsa-miR-193b*, hsa-miR-103/hsa-miR-138, hsa-miR-103/hsa-miR-198, hsa-miR-199b-3p/hsa-miR-139-3p, any one of hsa-miR-21/hsa-miR-139-3p and hsa-miR-21/hsa-miR-193b* or multiple nucleic acids combination.

7. each test kit of claim 1-6 is further used for hepatocellular carcinoma and healthy individual, colorectal cancer and lung cancer are differentiated.

8. the test kit of claim 7, wherein said expression of nucleic acid feature can comprise at least 16 kinds of nucleic acid molecule, preferably at least 6 kinds of nucleic acid molecule.

9. each test kit of claim 1-8, wherein said expression of nucleic acid feature comprise any one or Multi-encoding: Tumor-assaciated feature hsa-miR-122, hsa-miR-192, hsa-miR-215, hsa-let-7c, hsa-miR-103, hsa-miR-139-3p, hsa-miR-26b; The nucleic acid molecule of plasma specific feature hsa-miR-936, hsa-miR-193b*, hsa-miR-124, hsa-miR-34a, hsa-miR-198, hsa-let-7g, hsa-miR-363 and internal stability contrast has-miR-1228 and hsa-miR-1238.

10. the test kit of claim 9, wherein compare with one or more healthy individual, colorectal cancer and lung cancer, in described one or more target blood plasma, any one of coding hsa-miR-122, hsa-miR-192, hsa-miR-215, hsa-let-7c, hsa-miR-103, hsa-miR-26b, hsa-miR-34a, hsa-let-7g, hsa-miR-363 or multiple nucleic acids developed by molecule are raised; And any one or the multiple nucleic acids developed by molecule of coding hsa-miR-936, hsa-miR-193b*, hsa-miR124, hsa-miR-139-3p, hsa-miR-198 are reduced; Hsa-miR-1238 and hsa-miR-1228 do not change.

11. each test kit of claim 7-10, wherein said expression of nucleic acid feature comprises coding: hsa-miR-122/hsa-miR-936, hsa-miR-34a/hsa-miR-193b*, hsa-miR-34a/hsa-miR-198, hsa-miR-192/hsa-miR-936, hsa-miR-122/hsa-miR-193b*, hsa-miR-122/hsa-miR-198, hsa-miR-192/hsa-miR-124, hsa-miR-192/hsa-miR-193b*, hsa-miR-122/hsa-miR-124, hsa-miR-192/hsa-miR-198, hsa-miR-363/hsa-miR-936, hsa-miR-215/hsa-miR-193b*, hsa-miR-103/hsa-miR-936, hsa-miR-122/hsa-miR-139-3p, hsa-let-7c/hsa-miR-936, hsa-miR-215/hsa-miR-198, hsa-miR-192/hsa-miR-139-3p, hsa-miR-27b/hsa-miR-198, hsa-miR-26b/hsa-miR-936, hsa-let-7g/hsa-miR-936, hsa-miR-103/hsa-miR-198, hsa-let-7c/hsa-miR-193b*, hsa-miR-103/hsa-miR-193b*, hsa-miR-26b/hsa-miR-139-3p, hsa-let-7c/hsa-miR-198, hsa-miR-27b/hsa-miR-193b*, hsa-let-7g/hsa-miR-193b*, hsa-miR-363/hsa-miR-139-3p, hsa-let-7g/hsa-miR-198, hsa-miR-363/hsa-miR-198, hsa-miR-26b/hsa-miR-198, hsa-miR-103/hsa-miR-139-3p, hsa-miR-301a/hsa-miR-198, hsa-miR-26b/hsa-miR-193b*, hsa-let-7g/hsa-miR-139-3p, hsa-miR-363/hsa-miR-124, hsa-let-7c/hsa-miR-139-3p, hsa-miR-301a/hsa-miR-193b*, hsa-miR-301a/hsa-miR-139-3p, hsa-miR-26b/hsa-miR-124, hsa-let-7d/hsa-miR-198, hsa-let-7d/hsa-miR-139-3p, hsa-miR-103/hsa-miR-124, any one of hsa-miR-363/hsa-miR-193b* and hsa-let-7d/hsa-miR-193b* or multiple nucleic acids combination.

12. be used for differentiating the method for one or more target blood plasma that shows hepatocellular carcinoma, described method comprises:

(a) expression level of definite multiple nucleic acids molecule in described one or more target blood plasma, every kind of nucleic acid molecule encoding microrna sequences;

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

(c) expression level separately by contrasting in step (a) and obtaining (b), from described multiple nucleic acids molecule, identify one or more nucleic acid molecule of differential expression in described target blood plasma and contrast blood plasma, one or more nucleic acid molecule of wherein said differential expression represents each defined expression of nucleic acid feature such as claim 1-11 together, the existence of described expression of nucleic acid feature indication hepatocellular carcinoma.

13. the method for claim 12, it is further used for hepatocellular carcinoma and healthy individual, colorectal cancer and lung cancer are differentiated.

14. be used for the method for monitoring hepatocellular carcinoma treatment, described method comprises:

(a) by using method defined herein in one or more target blood plasma, to differentiate the expression of nucleic acid feature; And

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

15. be used for the method for prevention or treatment hepatocellular carcinoma, described method comprises:

(a) in blood, differentiate the expression of nucleic acid feature by the method for right to use requirement 12 or 13; And

The expression of one or more nucleic acid molecule of the coding microrna sequences that (b) the described expression of nucleic acid feature of change comprises in blood, described change is carried out in such a way, be that its expression of expressing the nucleic acid molecule that is raised in blood is reduced, and its expression of expressing the nucleic acid molecule of being reduced in blood is raised.

16. be used for preventing and/or treating the pharmaceutical composition of blood hepatocellular carcinoma, described composition comprises one or more nucleic acid molecule, every kind of equal encoding sequence of nucleic acid molecule,

Described sequence is with it expresses the coded at least part of complementation of microrna sequences of the nucleic acid molecule raised in from the blood plasma of patients with hepatocellular carcinoma as herein defined, and/or described sequence corresponding to as each defined coded microrna sequences of nucleic acid molecule that its expression is reduced in from the blood plasma of patients with hepatocellular carcinoma of claim 1-13.

17. the pharmaceutical composition of claim 16 is for the preparation of the purposes in the medicine that prevents and/or treats hepatocellular carcinoma.