CN102892898B

CN102892898B - For the diagnostic kit containing microRNA biomarker and the method for diagnosis of hepatoma

Info

Publication number: CN102892898B
Application number: CN201080064802.9A
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: 2016-03-09
Anticipated expiration: 2030-12-24
Also published as: CN102892898A

Abstract

The invention provides microRNA biomarker and comprise the diagnostic kit of nucleic acid molecule of microRNA biomarker described in Multi-encoding, for differentiating one or more target plasma showing hepatocellular carcinoma.Present invention also offers the method for the diagnosis and treatment of hepatocellular carcinoma, and for the pharmaceutical composition of prevention and therapy hepatocellular carcinoma.

Description

For the diagnostic kit containing microRNA biomarker and the method for diagnosis of hepatoma

Invention field

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

Background of invention

Hepatocellular carcinoma (also referred to as hepatocellular carcinoma (hepatocellularcarcinoma, HCC)) is one of modal solid malignant in world wide.Which represent the major histological type of liver cancer, the 70%-85% of all liver cancer cases may be accounted for.The world has about 500 every year, 000 new case, with the death of almost identical number, this reflects and lacks effective early detection and therapeutic choice (Thorgeirsson to this disease, S.S.andGrisham, J.W. (2002) Nat.Genet.31,339-346; Parkin, D.M.etal. (2005) CACancerJClin.55,74-108).

Therefore hepatocellular carcinoma is the cancer of a class poor prognosis.When patient's prognosis depends on that cancer is made a definite diagnosis by stages.Patients with hepatocellular carcinoma does not carry out the 5 years survival rate <5% performed the operation, and postoperative 5 years survival rates are 60%-70%.The row excision as tumor size <2cm, within 5 years, survival rate can reach 86%.But 3 years survival rate only 17%-21% of the capable any curer of early hepatocarcinoma patient (tumor size <5cm).This show cancer early find for treatment and survival of patients very important.(Tang,Z.Y.(2001)WorldJGastroenterol7,445-454;Chambers,A.F.etal.(2002)NatRevCancer2,563-572;Mola-KubaD.etal.(2006)AnnalsofHepatology5,16-24).

Although in recent years liver cell tumor morning find and excision significantly improve patient survival, most of tumour can not its emergence period and the non-lethal stage discovery.But only approximately the patients with hepatocellular carcinoma of 10%-20% meets surgical conditions at present, the surgical condition of described hepatocellular carcinoma is determined according to the relatively normal parameter such as liver function and accessible tumoral lesion by available different clinical staging systems.In addition, the patient carrying out surgical blanking has high-frequency transfer/recurrence usually, and postoperative 5 annual survival rates are only 30%-40%.Making a definite diagnosis of liver cancer relies on Histological Evidence usually.Tissues sampled is by puncture needle sucking-off or biopsy.But some liver cancer well differentiateds, that is they are made up of the liver cell of the maturation of almost growing completely, and therefore these liver cancer tissues exactly like non-cancer hepatic tissue under the microscope.And not all Pathology Doctors ' is all trained to distinguish WD liver cancer and just

Hepatic tissue puncture or the modal danger of biopsy are hemorrhage, especially because liver cancer is the tumour (holding many blood vessels) being rich in vascular.There are many cases may there is no need to carry out histodiagnosis by puncture or biopsy.If patient has Hazard Factor (such as liver cirrhosis, chronic hepatitis B or chronic hepatitis C) that liver cancer occurs and in blood, alfafoetoprotein (AFP) significantly improves, by biopsy, doctor almost just can not can determine that this patient there occurs liver cancer.At present, a kind of serum marker (a-alpha-fetoprotein, AFP) is only had to be generally used for early detection hepatocellular carcinoma (Mizejewski, G.J. (2003) ExpertRev.AnticancerTher.2,709-735; Paul, S.B.etal. (2007) Oncology72, Suppl.1,117-123).But this single labelledly has low specificity, and usually inappropriate due to false positive results.Serum AFP inspection only can be easy to detect liver cell tumor in the patient of 60%.On the other hand, in a large amount of liver cirrhosis patients, when there is not cancerous state, AFP also can raise.Therefore, we still need to determine some alternative molecule markers and the peripheral blood cell counts developing some sensitivities comes early discovery and differential diagnosis hepatocellular carcinoma.

A kind of approach addressed this problem can based on some little modulability RNA molecule, particularly based on microRNA (miRNA), they are little non-coding RNAs of the endogenous expression of a class evolution conservative, size is 20-25 Nucleotide (nt), can mediate the expression of said target mrna.Since they were found just to be considered to there is critical function in cell development, differentiation, proliferation and apoptosis before about 10 years.(Bartel,D.P.(2004)Cell116,281-297,Ambros,V.(2004)Nature431,350-355;He,L.etal.(2004)NatRevGenet5,522-531)。And miRNA is having superiority than mRNA as on cancer biomarkers because they in vitro very stable and also in vivo the life-span long.(Lu,J.etal.,(2005)Nature435,834-838;Lim,L.P.etal.,(2005)Nature433,769-773).

MiRNA produces from primary transcript, and primary transcript is processed as stem-ring structure precursor (pre-miRNA) by RNaseIIIDrosha.After transporte to cells matter, another kind is called that the RNaseIII of Dicer cuts the ring of pre-miRNA hair clip to form short double-strand (ds) RNA, and wherein a chain mixes in miRNA-protein (miRNP) as ripe miRNA.MiRNA instructs miRNP to arrive their said target mrna, and at this, they play function (Bartel, D.P. (2004) Cell23,281-292; He, L.andHannon, 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 mechanism that (RNAi) is identical by special cutting with RNA.Therefore, in this case, the function of miRNA is as short interfering rna (siRNA).Cell degradation approach is entered by guide or being translated property checks and do not affect mRNA level in-site with the complementary lower said target mrna of miRNA.But the mechanism how miRNA checks the translation of their said target mrna still has arguement.

High-throughout miRNA quantitative technique, such as miRNA biochip technology, the real-time TaqManmiRNA 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 and/or may develop relevant to the generation of some types of cancer.Such as, shown that two kinds of miRNA, miR-15 and miR-16-1 are positioned on the genetic loci of disappearance in chronic lymphatic leukemia (CLL), and found in the CLL patient of about 70%, two kinds of miR-96 gene are lacked or are lowered.In addition, in colorectum tumorigenesis, observe the downward of miR-143 and miR-145, and the expression of miRNAlet-7 often reduces (Michael, M.Z.etal. (2003) Mol.CancerRes.1,882-891 in lung cancer; Mayr, C.etal. (2007) Science315,1576-1579).In fact, based on miRNA express in cancer be correlated with change and miRNA be usually located at participate in cancer genome area observation supposition miRNA may both as tumor suppressor gene also as oncogene (Esquela-Kerscher, A.andSlack, F.J (2006) Nat.Rev.Cancer6,259-269; Calin, G.A.andCroce, C.M. (2007) J.Clin.Invest.117,2059-2066; Blenkiron, C.andMiska, E.A. (2007) Hum.Mol.Genet.16, R106-R113).The miRNA of unconventionality expression in these human cancer tissues is pointed out to highlight their potential as the biomarker of diagnosis and prognosis.

Some researchs report express spectra (Murakami, Y.etal. (2006) Oncogene25, the 2537-2545 of the miRNA in human hepatocellular carcinoma; Li, W.etal. (2008) IntJCancer123,1616-1622; Huang, Y.S.etal. (2008) Hepatology23,87-94; Ladeiro, Y.etal. (2008) Hepatology47,1955-1963; Jiang, J.etal. (2008) ClinCancerRes14,419-427).In addition, these researchs demonstrate, compared with nonmalignant liver cell or tissue, in pernicious cell or tissue, and specific miRNA unconventionality expression.Therefore, this type of miRNA can provide the understanding to the cell processes related in malignant transformation and progress.

In many possible sample types, blood is considered to optimal and monitors high-risk individuality, guiding early finds, early diagnosis, monitoring and Therapeutic cancer effectively because blood sample is easy to the method collection with Wicresoft.Being proved the miRNA that tumour derives is present in human plasma or serum with the form of quite stable, does not affect because they are protected by endogenous RNA enzymic activity.These are present in the level being derived from the miRNA of tumour in serum or blood plasma enough as the biomarker of cancer detection.And the miRNA in blood plasma or serum has strong dependency, shows no matter be that blood plasma or serum sample are all suitable for the biomarker of clinical application miRNA as cancer diagnosis.(Mitchell,P.S.etal.(2008)ProcNatlAcadSciUSA105,10513–10518;Gilad,S.etal.(2008)PLoSONE3,e3148;Chen,X.etal.(2008)CellRes18,997-1006)。So far, also do not determine in the blood plasma or serum of patients with hepatocellular carcinoma based on the miRNA biomarker of blood sample.

Therefore, be badly in need of the diagnostic flag based on blood, the particularly diagnostic flag of " expression characteristic (expressionsignature) " or " molecule footprint (molecularfootprint) " form, to make it possible to quick, reliable and cost-saving ground diagnosing hepatocellular carcinoma.And still need consistent method in high-risk individuals, to screen early hepatocyte cancer (earlystage-HCC), differential diagnosis HCC, early find liver cancer recurrence, and/or monitoring liver cancer treatment.

Purpose of the invention and overview

The object 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 blood, often kind of nucleic acid molecule is all encoded microRNA (miRNA) sequence, one or more of wherein said multiple nucleic acids molecule in the blood plasma of hepatocellular carcinoma by analysis compared with normal healthy controls and/or and healthy individuals, colorectal cancer compares differential expression with lung cancer, the nucleic acid molecule of one or more differential expression wherein said represents expression of nucleic acid feature together, the existence of this expression of nucleic acid feature instruction hepatocellular carcinoma, wherein said expression of nucleic acid feature comprises tumour correlated characteristic (tumor-relatedsignature) and plasma specific feature (plasma-specificsignature).

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

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

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

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

In preferred embodiments, described expression of nucleic acid feature comprises codes for tumor correlated characteristic hsa-miR-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 or the multiple nucleic acids molecule of contrast hsa-miR-1238 and hsa-miR-1228 of hsa-miR-629* and internal stability.

Particularly preferably, compared with one or more normal healthy controls, in one or more target plasma described: the expression of any one or the multiple nucleic acids molecule of coding hsa-miR-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 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 or the multiple nucleic acids developed by molecule of hsa-miR-629* are lowered, hsa-miR-1238 and hsa-miR-1228 does not change.

In a more preferred embodiment, expression of nucleic acid feature comprises any one or the multiple nucleic acids molecule of codes for tumor correlated characteristic 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, compared with one or more normal healthy controls, in one or more target plasma: one or more nucleic acid molecule any of coding hsa-miR-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 raised; And any one or the multiple nucleic acids developed by molecule of encode hsa-miR-139-3p, hsa-miR-193b*, hsa-miR-124 are lowered.

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

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

In particularly preferred embodiments, 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 or the multiple nucleic acids molecule of hsa-miR-21/hsa-miR-139-3p and hsa-miR-21/hsa-miR-193b*.

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

The expression of nucleic acid feature limited herein 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 one coding microrna sequences, and it is expressed to compare with one or more healthy individuals, colorectal cancer and lung cancer in one or more target plasma and is raised; And comprising the nucleic acid molecule of at least one coding microrna sequences, it is expressed to compare with one or more healthy individuals, colorectal cancer and lung cancer in one or more target plasma and is lowered.

In preferred embodiments, expression of nucleic acid feature comprises coding: tumour correlated characteristic 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 or the multiple nucleic acids molecule of has-miR-1228 and hsa-miR-1238.

Particularly preferably, compare with lung cancer with healthy individuals, colorectal 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 plasma is raised, and any one or the multiple nucleic acids developed by molecule of encode hsa-miR-936, hsa-miR-193b*, hsa-miR-124, hsa-miR-139-3p, hsa-miR-198 are lowered; Hsa-miR-1238 and hsa-miR-1228 does not change.

In a more preferred embodiment, expression of nucleic acid feature comprises any one or the multiple nucleic acids molecule of codes for tumor correlated characteristic 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 lung cancer with healthy individuals, colorectal cancer, any one or the multiple nucleic acids developed by molecule of encode in one or more target plasma hsa-miR-122, hsa-miR-192 and hsa-miR-215 are raised; And any one or the multiple nucleic acids developed by molecule of encode hsa-miR-936, hsa-miR-193b* and hsa-miR-124 are lowered.

In particularly preferred scheme, 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 or the multiple nucleic acids combination of hsa-miR-363/hsa-miR-193b* and hsa-let-7d/hsa-miR-193b*.

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) determines the expression level of multiple nucleic acids molecule in one or more target plasma described, often kind of nucleic acid molecule encoding microrna sequences; B () determines the expression level of described multiple nucleic acids molecule in one or more normal healthy controls blood plasma; C respective expression level that () is obtained in step (a) and (b) by contrast, one or more nucleic acid molecule of differential expression in described target plasma and contrast blood plasma is identified from described multiple nucleic acids molecule, one or more nucleic acid molecule of wherein said differential expression represents feature (signature) defined herein together, the existence of its instruction 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 plasma, and often kind of nucleic acid molecule is all encoded microrna sequences, and uses specific formulae discovery; B () determines the expression level that described nucleic acid molecule combines in one or more normal healthy controls blood plasma, and use specific formulae discovery; And (c) by compare the respective expression level obtained in (a) and (b) step differentiate described in be combined in difference in one or more target plasma described, wherein all representative features of combination of one or more differential expression, the existence of its instruction hepatocellular carcinoma.

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

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

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

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

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

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

Accompanying drawing is sketched

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

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

Fig. 3: the ROC tracing analysis (0: normal healthy controls group showing a routine hepatocellular carcinoma and the relevant miRNA of the tumour of two in normal healthy controls blood plasma (hsa-miR-122 and has-miR-139-3p); 1: hepatocellular carcinoma group).Result shows the Sensitivity and Specificity of these miRNA as diagnostic biomarker.These data obtained through microarray (biochip) analysis are standardized through internal stability contrast miR-1238.

Fig. 4: the mankind miRNA be included on the other hand in particularly preferred expression characteristic of the present invention is shown, differentiates hepatocellular carcinoma and healthy individuals, colorectal cancer and lung cancer with reference to application in long term of the present invention.Also show the expression level and accuracy (namely raise or lower) of as compared to healthy individuals, colorectal cancer and lung cancer the miRNA in patients with hepatocellular carcinoma.Be used as diagnostic biomarker in the combination of the first five miRNA and differentiate that the accuracy of hepatocellular carcinoma is at 85%-88%

Fig. 5: show by five kinds of different miRNA form four in the Comparison basis of combination.Between the data variation obtained by microarray and the data obtained by real-time quantitative PCR (quantitativeRT-PCR), quantitative dependency (R) is 0.76.These results show, and the miRNA feature obtained by AgilentmiRNA microarray (AgilentmiRNAmicroarray) is high confidence.

detailed Description Of The Invention

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

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

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

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

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

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

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

Term be defined in following use term further time provide.

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

An object of the present invention is 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 blood, often kind of nucleic acid molecule is all encoded microRNA (miRNA) sequence, one or more of wherein said multiple nucleic acids molecule in the blood plasma of hepatocellular carcinoma by analysis compared with normal healthy controls and/or and healthy individuals, colorectal cancer compares differential expression with lung cancer, the nucleic acid molecule of one or more differential expression wherein said represents expression of nucleic acid feature together, the existence of this expression of nucleic acid feature instruction hepatocellular carcinoma, wherein said expression of nucleic acid feature comprises tumour correlated characteristic and plasma specific feature.

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

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

The hepatocellular carcinoma of most common type is hepatocellular carcinoma (also referred to as liver cancer, being usually abbreviated as HCC).Term used herein " hepatocellular carcinoma " refers to the liver malignancy of former.Most of HCC is secondary to viral hepatitis infection (hepatitis B or hepatitis C) or liver cirrhosis (alcoholism is the most common factors causing liver cirrhosis).Be not in the right place in the country of disease in hepatitis, most of liver malignant cancer is not former HCC, but from the cancer metastasis (propagation) of other position of body such as colon.The therapeutic choice of HCC and prognosis depend on many factors, but depend on tumor size and by stages especially.Tumor grade (tumorgrade) is also important.High-grade (high-grade) tumor prognosis is not good, and inferior grade (low-grade) tumour also permits a lot of year out in the cold.Normally result is not good is because only the hepatocellular carcinoma of 10%-20% can be excised completely by operation.If cancer can not be excised completely, then this disease was fatal usually in 3-6 month.

Hepatocellular carcinoma is the same with other cancer any, occurs when existence causes cell high speed duplicating and/or causes the molecular mechanism of cells from apoptosis to be suddenlyd change.Especially, the chronic viral infection of hepatitis B and/or hepatitis C can help the generation of hepatocellular carcinoma by repeating to cause body self immune system to attack liver cell (some of them by virus infection, other be only onlooker).The mistake during although the constant circulation of repairing after this damage can cause repairing, cause carcinogenesis, current this hypothesis is more suitable for hepatitis C thereupon.But, in hepatitis B, be the most uniformly correlated factor in malignant tumour in the cell that virus genomic integration enters to infect.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 are not only considered to the Hazard Factor of tumor aetiology, but also be tumor development early stage/intergrade (i.e. " precancerous condition "), it is relevant to causing (being generally optimum) Non-Invasive excrescent excess proliferative tissue growth (can develop into malignant tumour as HCC) thereupon.

This malignant tumour is attacked other tissue and is often shifted when the time is enough.Malignant cell is characterised in that progressivity (progressive) and uncontrolled growth usually.Visual inspection HCC looks like nodositas or invasive carcinomas no.Nodular type tumour can be solitary (having agglomerate) or multiple (when developing into sclerosis complication).Tumor nodule is circular to oval, clear border but without coating.Diffuse type obscure boundary, and invade profit portal vein, seldom invade profit hepatic vein.

The mammalian target blood plasma adopted in the present invention can be people or nonhuman origin.But 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 plasma " refers at least assert it is display or the blood plasma with generation hepatocellular carcinoma tendency, and term " contrast blood plasma " typically refers to (health) type without this cancer phenotypic characteristic.But in some applications, such as, when comparing the blood plasma of display different carcinoma or precancerous condition, the cell with not too serious genius morbi is typically considered to " contrast blood plasma ".

Term used herein " blood plasma " is the yellow liquid component in blood, and the hemocyte in whole blood can normally suspend wherein.Its volume accounts for 55% of volume of whole blood.In blood plasma, most of composition is water (volume accounts for 90%), comprises soluble proteins, glucose, thrombin, mineral ion, hormone and carbonic acid gas (blood plasma is the main medium of movement transport).It is by fresh blood is centrifugal in whizzer that blood plasma prepares, until hemocyte sinks at the bottom of pipe.Then blood plasma is inclined to.The density of blood plasma is probably at 1025kg/m ³, or1.025kg/L.Recent research finds that miRNA is stable in blood plasma.Term " plasma sample " refers to the blood plasma to be checked obtained from individual or normal healthy controls.

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

Typically, target plasma used and contrast blood plasma are derived from from waiting to be diagnosed the biological sample collected in the object that whether there is hepatocellular carcinoma or hepatocellular carcinoma tendency occurs.In addition, in order to confirm the data of acquisition, " comparative sample " also can be collected from the object suffering from given known morbid state.Biological sample can comprise body tissue and liquid, as tissue, serum, hemocyte, phlegm and urine.In addition, if need, cell can from the body tissue obtained and liquid purifying, be then used 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 derivative

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

Term used herein " microRNA " (or " miRNA ") is its its ordinary meaning in this area (Bartel, D.P. (2004) Cell23,281-292; He, L.andHannon, G.J. (2004) Nat.Rev.Genet.5,522-531).Therefore, " microRNA " refers to the RNA molecule derived from genomic gene seat, and it is from the transcript processing that can form partial rna precursor miRNA structure.Ripe miRNA normal length is 20,21,22,23,24 or 25 Nucleotide, and the Nucleotide of other number also can exist, such as 18,19,26 or 27 Nucleotide.

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

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

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

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

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

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

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

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

In the scope of the invention, nucleic acid molecule is considered to differential expression, if the corresponding expression level of this nucleic acid molecule in target cell and compared with control cells typically differs at least 5% or at least 10%, preferably at least 20% or at least 25%, most preferably at least 30% or at least 50%.Therefore, the value of the latter raises at least 1.3 times or at least 1.5 times corresponding to the expression level of given nucleic acid molecule in target cell respectively compared to wild type control cells, otherwise or expression level in target cell lower at least 0.7 times or at least 0.5 times.

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

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

Standard program (the Sambrook set up well known in the art is typically followed in the determination of expression level, J.etal. (1989) MolecularCloning:ALaboraryManual.2ndEd., ColdSpringHarborLibraryPress, ColdSpringHarbor, NY; Ausubel, F.M.etal. (2001) CurrentPro-colsinMolecularBiology.Wiley & Sons, Hoboken, NJ).Determine to carry out at rna level, such as, use miRNA specific probe to carry out Northern engram analysis, or such as carried out at DNA level by quantitative PCR or real time pcr after reverse transcription (and clone) RNA group.Any nucleic acid molecule comprising the above-mentioned microrna sequences of analysis of encoding " determined " in term used herein.But, because pri-miRNA and the re-mRNA transformation period is short, typically only measure the concentration of ripe miRNA.

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

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

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

In the present invention, term " is identified hepatocellular carcinoma and/or differentiates other cancer types " and also comprising prediction and probability analysis (in " diagnosis " meaning).Composition disclosed herein and method are intended to clinical application, to determine form of therapy, comprise therapeutic intervention, and Case definition is as disease stage, and disease surveillance and surveillance of disease.According to the present invention, the intermediate result of check object state can be provided for.This intermediate result can combine to help doctor, nurse or other practitioner to diagnose out this object to suffer from this disease with extraneous information.Or the present invention can be used for the change being detected cancer tendency by blood sample, and provides useful information to doctor to diagnose.In addition, the present invention is also for distinguishing dissimilar liver cell tumor and other cancer types comprise colorectal cancer and lung cancer.

In the present invention, the nucleic acid molecule of one or more differential expression identified represents a kind of expression of nucleic acid feature together, and this expression of nucleic acid feature is the indication that there is hepatocellular carcinoma in target plasma.Term used herein " expression characteristic " refers to one group of nucleic acid molecule (such as miRNA), and wherein the expression level of each nucleic acid molecule contrasts between blood plasma different at (carcinous) target plasma and (non-cancerous).Herein, expression of nucleic acid feature also refers to a group echo and represents minimum object (difference) nucleic acid molecule, and often kind of nucleic acid molecule encoding can identify the miRNA sequence of the phenotypic status of target plasma.

In preferred embodiments, described expression of nucleic acid feature comprise at least one coding microrna sequences nucleic acid molecule, its express in one or more target plasma, contrast blood plasma with one or more compared with raised; And comprising the nucleic acid molecule of at least one coding microrna sequences, its expression is lowered compared with one or more contrasts

Term " plasma specific " (plasma-specific) herein refers to the feature of the variant expression of blood plasma at 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 be included in expression of nucleic acid feature is human sequence (hereinafter referred to as " has " (homo sapiens (Homosapiens))).

In a preferred embodiment of the invention, expression of nucleic acid feature comprises the relevant hsa-miR-122 (SEQIDNO:1) of codes for tumor, hsa-miR-199b-3p (SEQIDNO:2), hsa-miR-192 (SEQIDNO:3), hsa-miR-21 (SEQIDNO:4), hsa-miR-139-3p (SEQIDNO:5), hsa-miR-10a (SEQIDNO:6), hsa-miR-103 (SEQIDNO:7), hsa-miR-181d (SEQIDNO:8), hsa-miR-125b-2* (SEQIDNO:9), the expression of any one or the multiple nucleic acids molecule of hsa-miR-125a-3p (SEQIDNO:10), the hsa-miR-34a (SEQIDNO:11) of coding plasma specific, hsa-miR-136 (SEQIDNO:12), hsa-miR-151-5p (SEQIDNO:13), hsa-miR-193b* (SEQIDNO:14), hsa-miR-124 (SEQIDNO:15), hsa-miR-936 (SEQIDNO:16), hsa-miR-198 (SEQIDNO:17), hsa-miR-149* (SEQIDNO:18), hsa-miR-138 (SEQIDNO:19), hsa-miR-601 (SEQIDNO:20), hsa-miR-769-3p (SEQIDNO:21), hsa-miR-513c (SEQIDNO:22), hsa-miR-525-5p (SEQIDNO:23), hsa-miR-654-5p (SEQIDNO:24), hsa-miR-518c* (SEQIDNO:25), hsa-miR-500 (SEQIDNO:26), hsa-miR-181c* (SEQIDNO:27), hsa-miR-1226* (SEQIDNO:28), hsa-miR-202 (SEQIDNO:29), the expression of hsa-miR-629* (SEQIDNO:30) any one or multiple nucleic acids molecule, the hsa-miR-1238 (SEQIDNO:36) of encoding stable internal stability contrast and the nucleic acid molecule of hsa-miR-1228 (SEQIDNO:37).

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

table 1

All miRNA sequences disclosed herein have all been kept at (http://microrna.sanger.ac.uk/ in miRBase database; Also see Griffiths-JonesS.etal. (2008) Nucl.AcidsRes.36, D154-D158).

Particularly preferably, compared with contrasting in blood plasma with at one or more, encode hsa-miR-122 in one or more target plasma described, 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 or the multiple nucleic acids molecule of hsa-miR-151-5p is raised, and the hsa-miR-139-3p that encodes, 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 or the multiple nucleic acids molecule of hsa-miR-629* is lowered, hsa-miR-1238 and hsa-miR-1228 is unchanged.

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

In particularly preferred embodiments, adjust expression characteristic and comprise coding hsa-miR-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 or the multiple nucleic acids developed by molecule of hsa-miR-21/hsa-miR-139-3p and hsa-miR-21/hsa-miR-193b*.

Term used herein " Nucleic acid combinations " refers to and two or more nucleic acid expression level is integrally used.Particularly use dependency change or by same one-piece pattern calculation result.

In preferred embodiments, expression of nucleic acid feature comprises coding: tumour correlated characteristic hsa-miR-122 (SEQIDNO:1), hsa-miR-192 (SEQIDNO:4), hsa-miR-215 (SEQIDNO:31), hsa-let-7c (SEQIDNO:32), hsa-miR-103 (SEQIDNO:5), hsa-miR-139-3p (SEQIDNO:7), hsa-miR-26b (SEQIDNO:33); Any one or the multiple nucleic acids molecule of plasma specific feature hsa-miR-936 (SEQIDNO:16), hsa-miR-193b* (SEQIDNO:14), hsa-miR-124 (SEQIDNO:15), hsa-miR-34a (SEQIDNO:11), hsa-miR-198 (SEQIDNO:18), hsa-let-7g (SEQIDNO:34), hsa-30miR-363 (SEQIDNO:35) and internal stability contrast has-miR-1228 (SEQIDNO:36) and hsa-miR-1238 (SEQIDNO:37).

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

table 2

Particularly preferably, compare with lung cancer with in one or more healthy individuals, colorectal cancer, any one or the multiple nucleic acids developed by molecule of encode in one or more target plasma described 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 are raised; And any one or the multiple nucleic acids developed by molecule of encode hsa-miR-936, hsa-miR-193b*, hsa-miR-124, hsa-miR-139-3p and hsa-miR-198 are lowered; Hsa-miR-1238 and hsa-miR-1228 does not change.

In particularly preferred embodiments, 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 or the multiple nucleic acids combination of hsa-miR-363/hsa-miR-193b* and hsa-let-7d/hsa-miR-193b*.

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

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

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

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

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

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

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

Such as, at least one ribonucleotide main chain unit and at least one deoxyribonucleotide main chain unit may be comprised by this nucleic acid molecule.In addition, described nucleic acid molecule can be 2 '-O-methyl group or 2 '-O-methoxy group (methylating also referred to as 2 '-O-) containing one or more RNA backbone modifications, it prevents nuclease degradation in the medium, and be importantly also prevent the kernel of RNA inducibility silencing complex nuclease to be separated, cause the irreversible suppression of miRNA.Another possible modification (its function equivalence methylates in 2 '-O-) comprises locked nucleic acid (LNA), the nucleic acid analog of representative containing one or more LNA nucleotide monomer, described monomer is simulated in sugared conformation at RNA has locking bifuran sugar unit (Orom, U.A.etal. (2006) Gene372,137-141).

Be developed recently another kind of miRNA expression silencing gene.The chemical engineering oligonucleotide that these are called " antagomirs " is the RNA molecule (Krutzfeldt, J.etal. (2005) Nature438,685 – 689) of strand 23 Nucleotide puted together with cholesterol.As this chemically modified oligonucleotide another select, create as RNA produce from transgenosis can at the microRNA inhibitor of cells.These competitive inhibitors being called " microRNA sponge (microRNAsponges) " are the transcripts of expressing from strong promoter, multiple series combination site (Ebert containing interested microRNA, M.S.etal. (2007) Nat.Methods4,721-726).

In the particularly preferred embodiment of the inventive method, it is expressed and is selected from the microrna sequences as next group relative to expression characteristic by one or more nucleic acid molecule encoding lowered: hsa-miR-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 to comprise coding is imported in blood by the nucleic acid molecule of the microrna sequences of nucleic acid molecule encoding raised.In other words, the rise of the expression of the nucleic acid molecule of coding miRNA sequence realizes by being imported in one or more cell described by another copy (namely other " having justice " nucleic acid molecule) of described miRNA sequence.Described " having justice " nucleic acid molecule imported in one or more target cell can comprise the modification identical with above-mentioned " antisense " nucleic acid molecule.

In the particularly preferred embodiment of the inventive method, it is expressed and is selected from the microrna sequences as next group relative to expression characteristic by one or more nucleic acid molecule encoding raised: hsa-miR-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 and operably can be connected described nucleotide sequence is expressed with adjustment sequence with " having justice " and/or " antisense " nucleic acid molecule modifying the expression of the nucleic acid molecule of the microrna sequences comprised in one or more coding nucleic acid expression characteristic.

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

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

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

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

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

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

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

Within the scope of the present invention, suitable pharmaceutical composition comprises those compositions being suitable for oral, rectum, nose, locally (comprise through containing clothes and sublingual), peritonaeum and parenteral (comprising intramuscular, subcutaneous or intravenously) and giving, or by sucking or be blown into those compositions given.Can local or general give.Give preferably 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; such as capsule, micro-capsule, cachet, pill, tablet, powder, pilule (pellet), many granular preparations (such as pearl, particle or crystal), aerosol, sprays, foam, solution, dispersion agent, tincture, syrup, elixir, suspension, water-in-oil emulsion are as ointment, and water external emulsion is as emulsion, lotion and face cream.

Above-mentioned (" having justice " and " antisense ") nucleic acid molecule can be formulated as pharmaceutical composition (Gennaro by the use acceptable composition of pharmacology and the preparation method set up; A.L.andGennaro; A.R. (2000) Reming-n:TheScienceandPracticeofPharmacy; 20thEd.; LippincottWilliams & Wilkins; Philadelphia, PA; Crowder, T.M.etal. (2003) AGuide-PharmaceuticalParticulateScience.Interpharm/CRC, BocaRa-n, FL; Niazi, S.K. (2004) HandbookofPharmaceuticalManufacturingFormulations, CRCPress, BocaRa-n, FL).

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

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

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

A kind of method comprises passerby's chain of miRNA (miRNA* chain) and cholesterol or derivatives thereof/conjugate covalent coupling to promote by time at the absorption (Soutschek of the cell surface LDL receptors of expressing, J.etal. (2004) Nature432,173-178).Or the oligonucleotide (LNA-antimiR) that the locked nucleic acid of unconjugated PBS-preparation is modified may be used for general conveying (Elmen, J.etal. (2008) Nature452,896-899).The method of another kind of conveying miRNA comprises use polyoxyethylene glycol makes miRNA capsulation become specific liposome to reduce the absorption of scavenger cell and to strengthen cycling time.MiRNA is delivered to liver and (and does not arrive other organ (as Zimmermann by these specific nucleic acid particle (stable nucleic acid-lipid particle or SNALP) effectively; T.S.etal. described in (2006) Nature441,111-114)).In recent years, describe the agent delivery (Akinc of novel lipid sample delivery of molecules (synthesizing based on alkyl acrylate or alkyl-acrylamide and primary amine or the puting together addition of secondary amine) as RNAi therapeutical agent that a class is called lipidoids, A.etal. (2008) Nat.Biotechnol.26,561-569).

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

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

Embodiment

embodiment 1: sample collection and preparation

63 routine hepatic tissue excision samples.After collecting quick-frozen or collection in surgical operation sample liquid nitrogen, quick-frozen is immediately in liquid nitrogen.Sample is stored at-80 DEG C.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
HCC organizes	32
		Sum	63

Patient data's (age, sex, image data, treatment and other medical condition, family history like this) obtains and mates collected different samples from the database of hospital.Pathology follow (such as by h and E (H & E) dye the histologic analysis carried out) is for the consistent classification of the morbid state (i.e. contrast, precancer (such as liver cirrhosis), primary malignant neoplasm (such as hepatocellular carcinoma) or secondary/transfering malignant tumor (such as cancer embolus)) and guarantee sample of clearly determining given sample.

Detection wind lidar is optionally carried out with specific isolation tumor cell group (about 200000 cells) to each cancer sample.In brief, transparent transfer film is applied to the surface of tissue slice or sample.Under the microscope, observe the thin tissue section be placed on slide glass, and identification of cell group is to be separated.When the cell selected is positioned at the center of field of view, activate the integrated microscope optics of near-infrared laser diode (nearIRlaserdiodeintegralwiththemicroscopeoptics).Pulse laser beam activates the spot (spot) on transfer film, makes the cytogamy of this film and selection below.Then the transfer film with the cell of combination is peeled off (Emmert-Buck, M.R.etal. (1996) .Science274,998-1001 from described thin tissue section; Espina, V.etal. (2007) ExpertRev.Mol.Diagn.7,647-657).Substantially prepare freezing microtome section as manufacturers instructions and use laser capture microscope (ArcturusVeritas ^tMlaserCaptureMicrodissectionInstrument (MolecularDevices, Inc., Sunnyvale, CA, USA) carries out catching step.

Use manufacturer (Ambion, Austin, TX). mirVanamiRNA extraction agent box (mirVanamiRNAisolationkit) obtain total serum IgE.Total serum IgE A concentration is measured with NanoDrop1000 spectrophotometer (NanoDropTechnologies, Waltham, MA).Then RNA6000PicoLabChip test kit (AgilentTechnologies, SantaClara, CA) is used to realize by 2100Bioanalyzer to the quality monitoring of RNA.

embodiment 2; The analysis that in tissue samples, miRNA expresses

AgilentmiRNA microarray platform (AgilentTechnologies, SantaClara, CA, USA) is used optionally to carry out qualitative analysis to the miRNA that (difference) in specific sample is expressed according to manufacturers instructions.By the raw data normalization method of applying Quantile method and use R software known in the art will obtain for monochromatic (CY3) hybridization.

Data analysis aspect, the raw data obtained by monochromatic (CY3) applies the well-known Quantile method in this field and GeneSpringGX10 software (AgilentTechnologies, SantaClara, CA, USA) normalization method.Unpaired t-test (p<0.01) after Fisher inspection (F-test), is used for determining the differential expression of miRNA between normal liver tissue and HCC tissue.

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

embodiment 3: the collection of plasma sample and preparation

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

We collect blood sample 154 example of 2008-2009 cancer and healthy individuals 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.The blood sample of all patients obtains all before surgery.Patient data (age, sex, image data, treatment and other medical condition, family history like this) obtains from the database of hospital.The histopathologic classification of tumour is independently completed by three Pathology Doctors 's with reference to the World Health Organization (WHO) staging system.

the essential characteristic of table 4 blood preparation

The miRNA of genome range analyzes	Number
		Contrast
Healthy individuals	23
		Colorectal cancer	31
Lung cancer	36
		Hepatocellular carcinoma	24

		Quantitative RT-PCR is analyzed
Healthy individuals	16
		Hepatocellular carcinoma	24
Sum	154

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

MirVanaPARISmiRNA extraction agent box (mirVanaPARISmiRNAisolationkit) is used to obtain total serum IgE with reference to the operation instruction of manufacturer (Ambion, Austin, TX).Total rna concentration is measured with NanoDrop1000 spectrophotometer (NanoDropTechnologies, Waltham, MA).Then RNA6000PicoLabChip test kit (AgilentTechnologies, SantaClara, CA) is used to realize by 2100Bioanalyzer to the quality monitoring of RNA.

embodiment 4: miRNA expression pattern analysis in blood plasma

AgilentmiRNA microarray platform (AgilentTechnologies, SantaClara, CA, USA) is used optionally to carry out qualitative analysis to the miRNA that (difference) in specific blood product is expressed according to manufacturers instructions.This microarray takes from Sangerv.10.1 database, comprises 723 mankind miRNA probes.Total serum IgE (100ng) loading after Cy3 mark of each sample extracting in 114 blood.And XDRScan (PMT100, PMT5) scanning is obtained signal.The method of mark and hybridization is all with reference to AgilentmiRNA microarray specification sheets.

Data analysis aspect, internal reference (hsa-miR-1238) normalization method is stablized in the raw data obtained by monochromatic (CY3) application.Unpaired t-test (p<0.01) after Fisher inspection (F-test), to be used for determining between normal liver tissue and HCC tissue and or HCC relative to the differential expression of healthy individuals, colorectal cancer and lung cancer miRNA.

Use MedCalc software to HCC relatively and normal healthy controls and or HCC carry out recipient's performance characteristic relative to miRNA single in healthy individuals, colorectal cancer and lung cancer ((ROC) tracing analysis be to analyze the single Sensitivity and Specificity analysis of miRNA as diagnosing biomarker.Limiting significant credibility interval is 95%.

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

(i) differential expression >=2 times p value (probable value)≤0.05 changed; And

(ii) AUC (accuracy as diagnostic biomarker) >0.700

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

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

Show HCC shown in 5-7 below relative to normal healthy controls miRNA differential expression analysis data.Table 5 is listed in HCC and normal healthy controls to be organized and miRNA differential expression analysis in blood plasma.Table 6 to summarize in HCC and normal healthy controls miRNA differential expression analysis in blood plasma.Table 7 lists characteristic miRNA combination best in HCC patients blood plasma.The first five combined diagnosis biomarker be made up of six different miRNA to differentiate hepatocellular carcinoma and health accuracy at 87%-94%.In table hurdle, " t " represents HCC tissue, and " n " represents normal liver tissue, and " p " represents HCC patient, and " h " represents normal healthy controls.Particularly preferred miRNA (SEQIDNO:1-7 in table 5 and the SEQIDNO:11-17 in table 6) is also marked by with boldface type.

tumour in the blood plasma of table 5 hepatocellular carcinoma is correlated with miRNA feature

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

Second aspect differentiates hepatocellular carcinoma and healthy individuals, colorectal cancer and the preferred specific expressed data of lung cancer as shown in following table 8-10.The tumour that hepatocellular carcinoma listed by table 8 is correlated with miRNA expression characteristic; Table 9 shows plasma specific miRNA expression characteristic; And table 10 lists characteristic miRNA combination best in HCC patients blood plasma.The first five combined diagnosis biomarker be made up of six different miRNA is to differentiate that the accuracy of hepatocellular carcinoma and healthy individuals, colorectal cancer and lung cancer is at 85%-88%.In table hurdle, " t " represents HCC tissue, and " n " represents normal liver tissue, and " HCC " represents the blood plasma obtained from HCC patient, the blood plasma that " contrast " representative obtains from healthy individuals.Particularly preferred miRNA (SEQIDNO:1 in table 8, SEQIDNO:4, SEQIDNO:31; SEQIDNO:14-SEQIDNO:16 in table 9) also marked by with boldface type.

table 8 is for by relevant to the tumour that healthy individuals, colorectal cancer and lung cancer are distinguished for hepatocellular carcinoma miRNA expression characteristic

the plasma specific miRNA expression characteristic of table 9 for hepatocellular carcinoma and healthy individuals, colorectal cancer and lung cancer are distinguished

the combination miRNA expression characteristic of table 10 for hepatocellular carcinoma and healthy individuals, colorectal cancer and lung cancer are distinguished

embodiment 5: the checking of microarray data

In order to verify (with or quantitatively) data expressed of the miRNA that obtains of microarray, TaqManMicroRNA is adopted to analyze (AppliedBiosystems, FosterCity, CA, USA) real-time quantitative RT-PCR technology is with reference to its specification sheets (operation).Briefly, the TaqmanmicroRNARTKits of AppliedBiosystem is selected to illustrate that number carries out reverse transcription (RT) with reference to it.15ulRT system comprises, total serum IgE 10ng, 1X RT Buffer, 1XRT primer, 1nMdNTP, 4URNA enzyme inhibitors and 50UMultiScribe reversed transcriptive enzyme.Then RT system solution is above performed following program in PCR instrument (Thermalcycleralphaengine, Bio-rad): 16 ° of C, 30min; 42 ° of C, 30min; 85 ° of C, 5min.Quantitative PCR is then selected TaqManUniversalPCRMasterMix test kit and TaqmanmicroRNA to measure the test kits such as test kit and is illustrated with reference to it and uses.Quantitative PCR system comprises RTT product, 1XTaqManUniversalPCRMasterMix (NoAmpErase) UNG, 1XTaqManMicroRNAAssaymix of 2ul.Each reaction in triplicate.PCR in real time is carried out and is performed following program on RochLightCycling480: first 96 ° of C, 5min; Then 45 or 50 circulations, 95 ° of C, 15s; 60 ° of C, 60s.The method of Cp value second derivative (2ndderivative) completes on LC480 software.The Cp value of each miRNA is with stablizing internal reference hsa-miR-1228 normalization method.

The experimental data of the contrast platform of 4 miRNA combination of miRNA compositions as different in 5 in the blood plasma that Fig. 4 shows for deriving from 16 normal healthy controls and HCC patient.0.76 by microarray and the dependency of data multiple varied number that obtained by PCR in real time.The expression characteristic that the miRNA that AgilentmiRNA microarray obtains is described is high confidence.

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 herein represents unique miRNA expression characteristic, diagnosing hepatocellular carcinoma, not only can diagnosing liver cancer tendency state and also itself and colorectal cancer and lung cancer can be differentiated.

The miRNA characteristic identified in the present invention is expressed as to be screened with blood test, to find and diagnosing hepatocellular carcinoma provides distinctive molecule marker.And the expression of these characteristics is also used to monitoring 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 citing describe the present invention can suitably do not exist herein special disclose any element, restriction condition under carry out.Therefore, such as term " comprises ", " comprising " " to contain " etc. and should have broad sense and unrestricted.In addition; the term applied herein and express for describing the present invention and unrestricted meaning; and do not use these terms and express and get rid of any characteristic sum shown in it and describe or the meaning of Equivalent of its part, but should recognize can carry out various amendment in the scope of the invention of request protection.Therefore, although should understand the special announcement carried out the present invention by embodiment and optional feature, those skilled in the art can modify to the present invention and change, and this amendment and change are thought within the scope of the invention.

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

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

Claims

1. nucleic acid molecule is in the purposes shown in the diagnostic kit of one or more molecular marked compound in the blood of the target plasma of hepatocellular carcinoma for the preparation of discriminating, described nucleic acid molecule encoding microrna sequences,

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

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

The nucleic acid molecule of wherein said differential expression comprises the nucleic acid molecule of coding hsa-miR-193b*.

2. the purposes of claim 1, the nucleic acid molecule of wherein said differential expression comprises at least three ten two kinds of nucleic acid molecule.

3. the purposes of claim 1, the nucleic acid molecule of wherein said differential expression comprises at least six kinds of nucleic acid molecule.

4. the purposes of claim 1, the nucleic acid molecule of wherein said differential expression comprises the nucleic acid molecule of at least one coding microrna sequences, and its expression is raised in one or more target plasma with compared with contrasting in blood plasma; And comprising the nucleic acid molecule of at least one coding microrna sequences, its expression is lowered in one or more target plasma with compared with contrasting in blood plasma.

5. the purposes of any one of claim 1-4, the nucleic acid molecule of wherein said differential expression also 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 or the multiple nucleic acids molecule of contrast hsa-miR-1238 and hsa-miR-1228 of hsa-miR-629* and internal stability.

6. the purposes of claim 5, wherein compared with one or more normal healthy controls, in one or more target plasma described: the expression of any one or the multiple nucleic acids molecule of coding hsa-miR-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 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 or the multiple nucleic acids developed by molecule of hsa-miR-629* are lowered, hsa-miR-1238 and hsa-miR-1228 does not change.

7. profit requires the purposes of any one of 1-4, and the nucleic acid molecule of wherein said differential expression comprises any one or the multiple nucleic acids combination of coding hsa-miR-34a/hsa-miR-193b*, hsa-miR-122/hsa-miR-193b*, hsa-miR-192/hsa-miR-193b*, hsa-miR-103/hsa-miR-193b*, hsa-miR-10a/hsa-miR-193b*, hsa-miR-199b-3p/hsa-miR-193b* and hsa-miR-21/hsa-miR-193b*.

8. profit requires the purposes of 7, and the nucleic acid molecule of wherein said differential expression also comprises coding hsa-miR-21/hsa-miR-936, hsa-miR-192/hsa-miR-124, 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-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-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-122/hsa-miR-601, hsa-miR-21/hsa-miR-769-3p, hsa-miR-103/hsa-miR-138, hsa-miR-103/hsa-miR-198, any one or the multiple nucleic acids combination of hsa-miR-199b-3p/hsa-miR-139-3p and hsa-miR-21/hsa-miR-139-3p.

9. the purposes of any one of claim 1 or 4, wherein said test kit is further used for hepatocellular carcinoma and healthy individuals, colorectal cancer and lung cancer to differentiate.

10. the purposes of claim 9, the nucleic acid molecule of wherein said differential expression comprises at least 16 kinds of nucleic acid molecule.

The purposes of 11. claims 9, the nucleic acid molecule of wherein said differential expression comprises at least 6 kinds of nucleic acid molecule.

The purposes of 12. claims 9, the nucleic acid molecule of wherein said differential expression also comprises any one or Multi-encoding: tumour correlated characteristic 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-124, hsa-miR-34a, hsa-miR-198, hsa-let-7g, hsa-miR-363 and internal stability contrast the nucleic acid molecule of has-miR-1228 and hsa-miR-1238.

The purposes of 13. claims 12, wherein compare with lung cancer with one or more healthy individuals, colorectal cancer, in one or more target plasma described, any one or the multiple nucleic acids developed by molecule 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 are raised; And any one or the multiple nucleic acids developed by molecule of encode hsa-miR-936, hsa-miR-193b*, hsa-miR124, hsa-miR-139-3p, hsa-miR-198 are lowered; Hsa-miR-1238 and hsa-miR-1228 does not change.

The purposes of 14. claims 9, the nucleic acid molecule of wherein said differential expression 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 or the multiple nucleic acids combination of hsa-miR-363/hsa-miR-193b* and hsa-let-7d/hsa-miR-193b*.

The test kit of 15. any one of claim 1-14 is for the preparation of the purposes differentiating by the following method to show in the composition of one or more target plasma of hepatocellular carcinoma, and described method comprises:

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

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

C respective expression level that () is obtained in step (a) and (b) by contrast, one or more nucleic acid molecule of differential expression in described target plasma and contrast blood plasma is identified from described multiple nucleic acids molecule, one or more nucleic acid molecule of wherein said differential expression represent together as any one of claim 1-14 the expression of nucleic acid feature that defines, the existence of described expression of nucleic acid feature instruction hepatocellular carcinoma.

The purposes of 16. claims 15, it is further used for hepatocellular carcinoma and healthy individuals, colorectal cancer and lung cancer to differentiate.

17. the test kit of any one of claim 1-14 for the preparation of monitor by the following method hepatocellular carcinoma treatment composition in purposes, described method comprises:

A method that () is defined by use claim 15 or 16 differentiates expression of nucleic acid feature in one or more target plasma; And

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