CN1659287A - Methods of diagnosing potential for metastasis or developing hepatocellular carcinoma and of identifying therapeutic targets - Google Patents

Abstract

The present invention relates to methods for diagnosing the metastatic potential of hepatocellular carcinoma (HCC) in HCC patients and methods for diagnosing the potential of developing HCC in patients with chronic liver diseases. A computer readable medium, a digital computer, and a system useful for such diagnosis are also provided. Further disclosed are methods for identifying potential therapeutic targets for treating metastasis in HCC patients and methods for preventing HCC in patients with chronic liver diseases. In addition, the invention provides methods for inhibiting metastasis in HCC patients by suppressing the function of one therapeutic target, osteopontin, and methods for preventing the development of HCC in patients with chronic liver diseases by suppressing the function of one therapeutic target, EpCAM. Pharmaceutical compositions containing agents capable of inhibiting the functions of osteopontin or EpCAM are also disclosed.

Description

The method of diagnosing liver cancer transfer or initiation potential and evaluation treatment target spot

The cross reference relation of related application

This application requires the U.S. Provisional Patent Application No.60/370 of submission on April 5th, 2002,895 right of priority, and the full content of this application is introduced into this paper as a reference.

To the research of federal government-funded or the power statement of the following invention of finishing of exploitation

The present invention returns United States Government's (is representative with healthy and the human secretary of service department) to own.

Background technology

Hepatocellular carcinoma (HCC) is one of the most common and invasive malignant tumour of tool in the world wide, and curative ratio is lower than 5%.The major cause that mortality ratio is high is that shifting appears in cancer cells in liver.People know little about it to the molecular mechanism that shifts in the relevant liver or this type of patient's specific treatment target spot.

In in the past 10 years, many technology make the expression level of a large amount of transcriptons (transcripts) of a monitoring time point in office become possiblely (for example to see people such as Schena, Science 270:467-470,1995; People such as Lockhart, NatureBiotechnology 14:1675-1680,1996; People such as Blanchard, Nature Biotechnology 14:1649,1996; With U.S. Patent No. 5,569,588).For the organism of knowing the complete genome group, all gene transcription are possible in the analysis of cells.For the other biological body,,, might in cell, monitor a large amount of genes simultaneously along with understanding gradually to human genome information as the people.These Monitoring techniques generally are applied to identify the gene that raises or reduce under different pathological or physiological status, be applied to analyze the member of transfer cell status signal, and be applied to identify various drug targets.

The inventor has analyzed 9180 expression of gene in the HCC tissue, and pathological tissue is from being attended by cancer metastasis in the liver or not following 40 patients of transfer.Use supervision machine learning algorithm (supervised machinelearning algorithm) that patient's allelic expression is classified, produced a part signal characteristic (signature) first, this signal characteristic can correctly shift the patient classification with shifting patient and non-, and has identified and the more closely-related genes of prognosis (comprising patient's survival rate).Genetic expression signal with the genetic expression signal of former the HCCs focus that shifts and corresponding metastatic lesion is closely similar, and this shows that the gene that helps spreading starts in primary tumo(u)r.And in the primary HCC that in liver, shifts, the osteopontin overexpression, and external intrusion experiment shows that the neutralizing antibody of anti-osteopontin can stop the intrusion of high secondary liver cancer cell.These data show that osteopontin both can be used for diagnosis, can be used as the treatment target spot of transitivity HCC again.

This research is analyzed 9180 expression of gene in the tumor sample, and sample has serious hepatopathy (contain highly dangerous develops into HCC and low degree of hazard develops into HCC two classes) from 54 HCC patients' tumor tissues and 59 but the liver organization of canceration not.High-risk group comprises hepatitis B, third liver, hemochromatosis and WilsonShi patient.Low danger group comprises alcoholic liver disease, autoimmune hepatitis and primary biliary cirrhosis.Between high-risk group and the low danger group gene expression dose identify one group of important function of gene more, can come high-risk group and low danger group are distinguished with these genes.Use and from the expression data of HCC sample this is organized remarkable gene and filter, identified and have feature and the subgroup gene that can be used for sample classification of multiple HCC associated molecule.In addition, EpCAM is as one of the most remarkable gene, and its overexpression is proportionate with the danger of serious liver problem sufferer's secondary HCC, suppresses its expression the growth of HCC cell is suppressed.Therefore, EpCAM is the diagnostic markers that prediction develops into HCC danger, also is simultaneously to stop chronic hepatitis patients to develop into the treatment target spot of HCC.

Summary of the invention

One aspect of the present invention relates to a kind of method, and this method can be identified the potential treatment target spot that suppresses transfer for HCC patient; For chronic hepatitis patients, can stop and develop into HCC.

A kind of method that suppresses the potential treatment target spot that HCC patient shifts of identifying may further comprise the steps: a) will contact with the chip that comprises at the capture agent of one group of cell sign thing from transitivity HCC patient's sample; B) from sample, catch mark and produce first signal; C) thus with the HCC patient's of non-transfer sample repeating step a) and step b) produce second signal; D) compare first and second signals, thereby identify the different cell sign thing subgroup of level of first signal and second signal, the cell marker of this subgroup is exactly the potential treatment target spot that treatment HCC shifts.In some specific embodiment, the signal that the normal non-cancer tissue sample of deduction is produced on chip (identical with the chip of step a)) in step b) and step c), thus produce first and second signals.

A kind of discriminating prevents that chronic hepatitis patients from developing into the method for the potential treatment target spot of HCC, may further comprise the steps: a) will contact with the chip that comprises at the capture agent of one group of cell sign thing from the sample of the high-risk chronic hepatitis patients of HCC; B) from sample, catch mark and produce first signal; C) a) and step b), with the chronic hepatitis patients sample repeating step of the low danger of HCC thus produce second signal; D) compare first and second signals, thereby identify the different cell sign thing subgroup of level of first signal and second signal, the cell marker of this subgroup is exactly to prevent that chronic hepatitis patients from developing into the potential treatment target spot of HCC.In some specific embodiment, the signal that the normal non-cancer tissue sample of deduction is produced on chip (identical with the chip of step a)) in step b) and step c), thus produce first and second signals.

The present invention relates to prediction HCC patient's metastatic potential on the other hand or predicts that chronic hepatitis patients develops into the method for the danger of HCC.

A kind of method of predicting HCC patient's metastasis of cancer possibility, comprise the steps: a) and will contact with the chip that comprises at the capture agent of one group of cell sign thing that this group cell sign thing comprises at least 10 genes independently selecting or the albumen of coded by said gene from table 2 gene from transitivity HCC patient's sample; B) from sample, catch mark; C) from the mark that is hunted down of step b), produce first signal; D) thus a) produce second signal to step c) with the HCC patient's of non-transfer sample repeating step; E) thus shift possible HCC patient's sample repeating step and a) produce the 3rd signal to step c) with indeterminate having or not; F) the 3rd signal and first, second signal are compared, thereby whether the HCC patient of clear and definite step e) has the transfer possibility.In some specific embodiment, the cell sign thing that carries out above-mentioned experiment comprises at least 20, preferably 50, more preferably 100, and whole best gene or by the albumen of genes encoding, these genes are independently selected from table 2.In other specific embodiments, this group cell sign thing comprises from the gene of table 4 or the albumen of coded by said gene, or is numbered the single-gene (Unigene) of Hs.313, Hs.69707, Hs.222, Hs.63984, Hs.75573, Hs.177687, Hs.69707, Hs.222, Hs.323712 and Hs.63984.Preferably, step a), b), d), e) specimen in use is preferably the hepatic tissue extract.In a preference, the chip of step a) is a genome chip.In another preference, the chip of step a) is the protein groups chip.

A kind of chronic hepatitis patients of predicting develops into the method for the danger of HCC, may further comprise the steps: a) will contact with the chip that comprises at the capture agent of one group of cell sign thing from the sample of the high-risk chronic hepatitis patients of HCC, this group cell sign thing comprises the albumen (by independently selecting in table 5 gene) of 10 genes or coded by said gene at least; B) from sample, catch mark; C) from the captive mark of step b), produce first signal; D) thus a) produce second signal to step c) with the chronic hepatitis patients sample repeating step of the low danger of HCC; E) thus a) produce the 3rd signal to step c) with the chronic hepatitis patients sample repeating step of the indeterminate HCC of having or not danger; F) the 3rd signal and first, second signal are compared, thus determining step e) the patient develop into the danger of HCC.In some specific embodiment, the cell sign thing that carries out above-mentioned experiment comprises at least 20, preferably 50, more preferably 100, whole gene or the albumen of coded by said gene best, and these genes are independently selected from table 5.Sometimes, the cell sign thing is from the gene of table 6 or table 7 or the albumen of coded by said gene.Preferably, step a), b), d), e) specimen in use is the hepatic tissue extract.In a preference, the chip of step a) is a genome chip.In another preference, the chip of step a) is the protein groups chip.In certain embodiments, the patient who has highly dangerous to develop into HCC suffers from hepatitis B infected, third liver, hemochromatosis and WilsonShi disease.In other examples, develop into the lower patient of HCC danger and suffer from alcoholic liver disease, autoimmune hepatitis and primary biliary cirrhosis.In other examples, the patient of the risk level of trouble HCC to be assessed suffers from hepatitis B, third liver, hemochromatosis, WilsonShi disease, alcoholic liver disease, autoimmune hepatitis or primary biliary cirrhosis.

Another aspect of the present invention relates to the method that suppresses HCC patient's metastasis of cancer and suppresses the method that chronic hepatitis patients develops into HCC.The method that suppresses HCC patient's metastasis of cancer comprises step: the activity that suppresses OPN.In certain embodiments, can suppress the activity of OPN, be preferably and adopt the special antisense polynucleotides of OPN by the expression that suppresses OPN.In addition, can combine the activity that suppresses OPN with specificity between its acceptor, be preferably the anti-OPN antibody of employing by suppressing OPN.Prevent that the method that chronic hepatitis patients develops into HCC from comprising step: the activity that suppresses EpCAM.In certain embodiments, can suppress the activity of EpCAM, be preferably and adopt special antisense polynucleotides of EpCAM or little interferential RNA by the expression that suppresses EpCAM.In addition, can combine the activity that suppresses EpCAM with specificity between its acceptor, be preferably employing anti-EpCAM antibody by suppressing EpCAM.

Of the present invention relating in one aspect to again is used to assess HCC patient's cancer metastasis possibility or assesses computer-readable medium, digital computer and the system that chronic hepatitis patients develops into HCC danger.

The computer-readable medium of assessment HCC patient cancer metastasis possibility comprises: a) code of first data set, this data set derives from first signal, the chip that this signal contacts from the sample with transitivity HCC patient, described chip comprises the capture agent at one group of cell sign thing, and this group cell sign thing comprises the albumen (by independently selecting in table 2 gene) of at least 10 genes or coded by said gene; B) code of second data set, this data set derives from second signal, the chip that this signal contacts from the sample with non-metastatic HCC patient, described chip is identical with a) chip; C) code of the 3rd data set, this data set derives from the 3rd signal, the chip that this signal contacts from the HCC patient's who shifts with the unknown sample, described chip is identical with a) chip; D) code that the 3rd data set and first and second data sets are compared.A kind of digital computer also is provided, and it comprises the described computer-readable medium that is used for assessing HCC patient's cancer metastasis possibility.A system also is provided, and it comprises such digital computer, comprise at the chip of the array of the capture agent of one group of cell sign thing (described mark comprise at least 10 genes independently selecting from table 2 gene or the albumen of coded by said gene) and can read the reader of signal from chip contacting the back with sample.

The computer-readable medium that the assessment chronic hepatitis patients develops into HCC danger comprises: a) code of first data set, this data set derives from first signal, the chip that this signal contacts from the patient's high-risk with suffering from chronic hepatopathy and HCC sample, described chip comprises the capture agent at one group of cell sign thing, and this group cell sign thing comprises the albumen (by independently selecting in table 5 gene) of at least 10 genes or coded by said gene; B) code of second data set, this data set derives from second signal, and this signal is from the chip that contacts with the patient's of the low danger of chronic hepatopathy and HCC sample, and described chip is identical with a) chip; C) code of the 3rd data set, described data set derives from the 3rd signal, and this signal is from chronic hepatopathy and develop into the chip that patient's the sample of risk level the unknown of HCC contacts, and described chip is identical with a) chip; D) code that the 3rd data set and first and second data sets are compared.Digital computer also is provided, and it comprises and is used for assessing the described computer-readable medium that chronic hepatopathy develops into the danger of HCC.A system also is provided, and it comprises such digital computer, comprise at the chip of the array of the capture agent of one group of cell sign thing (described mark comprise at least 10 genes independently selecting from table 5 gene or the albumen of coded by said gene) and can read the reader of signal from chip contacting the back with sample.

Definition

Except as otherwise noted, Science and Technology term used herein is the common meaning of understanding of this field that the present invention belongs to professional person.Following document provides the General Definition of used many terms among the present invention: people such as Singleton, Dictionary of Microbiology and Molecular Biology (the 2nd edition, 1994); The CambridgeDictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, the 5th edition, people such as R.Rieger (eds.), Springer Verlag (1991); With Hale ﹠amp; Marham, The Harper CollinsDictionary of Biology (1991).As used herein, except as otherwise noted, following term has the implication under it.

As used herein, term " hepatocellular carcinoma " or " HCC " refer to account for the liver cancer of the main type more than 90% of primary hepatocarcinoma.The liver cancer cell state comprises from well differentiated to the height degeneration and undifferentiated damage.Liver cancer can shift outward for transfer or liver in pathology (non-transfer), the multiple liver in single focus liver.

" high-risk disease before the cancer " refers to the disease of one group of epidemiology definition, and these diseases have highly dangerous to develop into HCC.These diseases comprise chronic viral hepatitis B, third liver, hemochromatosis and WilsonShi disease.

" low danger disease before the cancer " refers to the disease of one group of epidemiology definition, and these diseases have low degree of hazard to develop into HCC.These diseases comprise alcoholic liver disease, autoimmune hepatitis and primary biliary cirrhosis.

Term " transfer " or " transfer " refer to single cancer cell infiltration (intrusion) surrounding tissue, enter the recycle system and in the ability of new position neoplasm.

" non-transfer " refers to that tumour is not diffused into beyond the primary lesion, and refers to that particularly it does not enter the recycle system and in new position neoplasm.

Term " non-cancer " refers to biology or tissue sample; cell wherein is normal or non-pathomorphism, can adopt naked eyes, analyze on molecular level by the antibody or the nucleic acid probe of microscope, immunohistology, immunology or applying detection pathological condition.

Term " normally " refers to never to suffer from biological sample or the tissue sample that the individuality of any disease of disease collects before disease before HCC, the high-risk cancer, the low danger cancer.

Term " capture agent " refer to arbitrary can with specific nucleic acid or protein marker bonded material.Typically, can control by the condition in the cohesive process and have combining of mark and capture agent.As, the nucleic acid mark can be by used hybridization conditions control with combining of oligonucleotide.The nucleic acid mark that strict hybridization conditions only allows high homology (as with oligonucleotide the homology of 95%-100% being arranged) combines with oligonucleotide.

" array " refers to be incorporated into a plurality of capture agents of substrate (as solid support), and these capture agents can be incorporated into relevant mark.For example, array can be made up of nucleic acid molecule, protein molecular or other reagent, can be specifically in conjunction with isolating nucleic acid, albumen or polypeptide from biological sample.Capture agent when the correlating markings thing is incorporated into capture agent, can be determined binding capacity so preferentially with the addressable mode combination.

" dna microarray " refers to that capture agent is the array of nucleic acid molecule.Typically, the DNA array is made up of the DNA oligonucleotide of certain-length, under certain condition can with DNA, cDNA or RNA molecular hybridization.The DNA oligonucleotide can be the short-movie section Nucleotide of 15～50 bases, also can be to be 500～1000 bases or the Nucleotide of long segment more.Dna microarray can be made up of hundreds of or thousands of different nucleic acid molecule, and each nucleic acid molecule is in the fixed position on array.But when mark by behind the detection molecules mark, combining usually of mark and dna microarray can be by quantitatively.Term dna microarray and term " genome array " are used interchangeably.

" protein array " refers to the array of capture agent energy conjugated protein mark.Typically, capture agent is polyclone or monoclonal antibody, can with special protein bound.In other words, any albumen, polypeptide, nucleic acid or other molecules or surface that can binding proteins specific can be applied in the protein arrays.These arrays generally include the hundreds of or thousands of different capture agents that are positioned at addressable area.But when mark by behind the detection molecules mark, the combining usually by quantitatively of the capture agent on the protein array and mark.Term protein array and term " protein group array " are used interchangeably.

" gene expression profile " refers to compare the gene that all are expressed with standard model in organizing sample.Expression of gene level in the gene expression profile can be determined by the expression level of standard of comparison sample and sample to be checked such as HCC tumor sample or serious liver problem sufferer's sample.Be used for determining the standard model of HCC metastases possibility, for non-cancer liver organization or be the patient's that is not diagnosed as HCC liver organization.Be used for determining that serious liver problem sufferer develops into the standard model of HCC possibility, be the patient's that is not diagnosed as serious hepatopathy liver organization.Compare with standard model, the gene in the sample to be checked may be crossed and express or low the expression.

" transitivity genetic expression predictor (predictor) " refers to the expression of the cluster specific gene relevant with the diagnosis of transitivity HCC.Transitivity genetic expression predictor can draw like this: more non-transfer HCC sample and and the gene expression profile of the HCC sample that shifts, use a clear and definite algorithm or one group of algorithm to carry out cluster analysis and classification analysis then.The gene number can be with the parameter in the used cluster algorithm exclusive disjunction rule (as p-level=0.001vs.0.022) change.

" HCC genetic expression predictor " refers to may develop into diagnosis the expression of the relevant cluster specific gene of the patient of HCC.HCC genetic expression predictor can draw like this: the gene expression profile of non-metastatic liver sample that develops into the high-risk patient of HCC and the non-metastatic liver sample that the patient who develops into the low danger of HCC is arranged is relatively arranged, use a clear and definite algorithm or one group of algorithm to carry out cluster analysis and classification analysis then.The gene number can be with the parameter in the used cluster algorithm exclusive disjunction rule (as p-level=0.001vs.0.022) change.

Table 2-7 used " UG bunch " refers to the UniGene database by national bioinformation center (NCBI) editor.Each accession number in the UniGene database is the compilation of all Nucleotide and amino acid sequence data, can be used for a specific nucleotide sequence.Can provide and the linking of GeneBank or other databases as, each UG bunch accession number, the latter can provide the part of encoding gene or the nucleotide sequence of full-length cDNA.In other words, link can provide genome or est sequence data or amino acid sequence information.Each UG bunch accession number provides unique sequence information for specific gene, nucleic acid or the aminoacid sequence of being identified.

" osteopontin " refers to that its Genebank accession number is NM_000582 by secretor type phosphoprotein or its conservative variations thing of SEQ ID NO:1 coding.Also can find its nucleic acid and amino acid sequence information at the UniGene of NCBI database, its accession number on the NCBI network address is Hs.313.The NCBI network address has been listed 9 mRNA/ genomic dna sequences and 900 above expressed sequence tag (EST).Osteopontin is an extracellular protein, and is relevant with ground substance of bone and atherosclerotic plaque.The osteopontin of total length comprises a RGD aminoacid sequence, and this RGD aminoacid sequence is the binding site of integrin.Osteopontin is the main part of glass connection protein receptor." OPN " can exchange with osteopontin and use, all the gene of finger protein, proteins encoded or its fragment.

" EpCAM " is the glycoprotein of a kind of 40kDa, and function is an epithelial cell adhesion molecule.Be confirmed as the calcium ion signal transducer (being also referred to as TACSTD1) with tumour, UniGene bunch of accession number is Hs.692, and EpCAM is by gene GA733-2 coding, and this gene is positioned on the human chromosomal 4q.EpCAM is the transmembrane protein of expressing in the cell of epithelial origin, can mediate the adhesion between the homotype cell that does not rely on calcium ion, can be by many known monoclonal antibody specific recognition, as 17-1A, 323/A3, KS1/4, GA733, MOC31 etc.

Term among the present invention " mark " refers to the nucleotide sequence or the gene of coded polypeptide (specific apparent molecular weight is arranged); it is transitivity HCC patient or easily to suffer from the HCC individual sample in the respective sample with contrast individual (as non-metastatic HCC patient, the individuality that is not diagnosed as cancer or do not detect cancer, normal or healthy people) be that otherness exists.Mark also can refer to polypeptide or the protein by nucleotide sequence or genes encoding; it is transitivity HCC patient or easily to suffer from the HCC individual sample in the respective sample with contrast individual (as non-metastatic HCC patient, the individuality that is not diagnosed as cancer or do not detect cancer, normal or healthy people) be that otherness exists.Mark among the present invention comprises gene and its encoded protein matter of hereinafter showing to have among the 2-7 UG bunch of accession number.

As used herein, term " sample " refers to biological organization or tissue juice sample, and they can be used for determining the source of gene expression profile, mark or comprise relevant albumen (as osteopontin or EpCAM) or this proteic nucleic acid of coding.Such sample comprises that (but being not limited to) from various types of tissues that the human body separation comes, also can comprise tissue slice such as freezing microtome section or paraffin section.Tissue comprises liver sample and humoral sample (blood, serum, blood plasma, urine and other body fluid.The used preferred sample of the present invention is the cell lysates that obtains from tissue of interest (as liver) extracting, such cell lysates can prepared in various methods well known to those skilled in the art, this depends on the form of to be detected and the cell sign thing checked, as nucleic acid (as mRNA) but, albumen or molecule with other detection of biological characteristics (as enzymic activity).

Those have and regulate for the compound resemble important protein biological activities such as osteopontin or EpCAM for assay determination, the term that context is mentioned " functional effect " should comprise the correlation parameter that mensuration is influenced by OPN or EpCAM indirectly or directly, for example the mRNA level of proteins encoded, protein level and they are on function, and effect (their binding substancess natural for example of physics and chemistry with it, for example other albumen, nucleic acid or other molecules carry out the interactional ability of specificity; And a series of active abilities of their conditioning signals transduction causing cell, for example cell increment, differentiation, apoptosis, secretion, adhesion etc.).

" nucleic acid " refers to thymus nucleic acid or Yeast Nucleic Acid and polymkeric substance thereof, can be strand or double chain form.This term should include the nucleic acid that contains the known nucleic acid analogue or basic framework residue by the nucleic acid of modified or as the nucleic acid that connects thing, they can be synthetic, exist naturally and non-existence naturally, compare with contrast nucleic acid, they have similar binding characteristic, and be similar to the contrast Nucleotide mode by metabolism.The example of these analogues has methylphosphonate, 2-O-methyl ribonucleotides, the peptide nucleic acid(PNA) (PNAs) of thiophosphatephosphorothioate, phosphoramidate, methylphosphonate, chirality.This term also comprises the nucleic acid of separation acquisition from biological sample and the oligonucleotide of synthetic.

Unless otherwise indicated, a specific nucleotide sequence also impliedly comprises its conservative nucleic acid varient (for example degenerate codon replacement) and their complementary sequence of modifying, and also comprises the sequence of expressing.Particularly, the replacement of degenerate codon can obtain by formation sequence, in the sequence of this generation, the 3rd the mixed base in position and/or the Hypoxanthine deoxyriboside residue of one or more selected (or all) codons replaces (people such as Batzer, Nucleic AcidRes.19:5081,1991; People such as Ohtsuka, J.Biol.Chem.260:2605-2608,1985; People such as Rossolini, Mol.Cell.Probes 8:91-98,1994).This term of nucleic acid can exchange with gene, cDNA, mRNA, oligonucleotide and polynucleotide and use.

Term " polypeptide ", " peptide " reach " albumen " and are used interchangeably at this paper, refer to the polymkeric substance of amino-acid residue.This term can refer to such aminoacid polymers, and wherein one or more amino-acid residues are to the corresponding natural amino acid whose artificial chemical simulation thing that exists, and also can refer to the aminoacid polymers that naturally occurring aminoacid polymers and non-natural exist.

Term " amino acid " refers to the amino acid of naturally occurring and synthetic, and with the amino acid analogue and the amino acid analog thing of naturally occurring amino acid performance same function.Naturally occurring amino acid is by genetic code amino acids coding and those adorned afterwards amino acid, for example oxyproline, Gla and O-phosphoserine.Amino acid analogue refers to these compounds, they have with the identical basic chemical structure of naturally occurring amino acid, for example be incorporated into alpha-carbon atom, carboxyl, amino and the R base of H, for example homoserine, nor-leucine (glycoleucine), methionine sulfoxide, methionine(Met) methyl sulfonium.These analogues have the R group (as nor-leucine) of modified or the polypeptide backbone of modified, but have kept the basic chemical structure identical with natural amino acid.The amino acid analog thing is meant such chemical compound, and their structure is different from amino acid whose general chemical structure, but on function with natural amino acid similarity.

The three-character doctrine that amino acid can be recommended by they common known IUPAC-IUB commission on Biochemical nomenclatures, or represent by monocase.Equally, nucleic acid can be represented by received one-letter code usually.

" the conservative varient of modifying " all is suitable for aminoacid sequence and nucleotide sequence.For specific nucleotide sequence, the conservative varient of modifying refer to the to encode nucleic acid of identical or identical in essence aminoacid sequence does not perhaps refer to identical in essence sequence during encoding amino acid sequence when nucleic acid.Because the degeneracy of genetic code has the arbitrary specific albumen of the identical nucleic acid codified of a large amount of functions.For example, codon GCA, GCC, GCG and the GCU L-Ala of all encoding.Therefore, in each position that L-Ala is limited by a codon, this codon can become in the above-mentioned corresponding codon any and can not change the polypeptide that is encoded.Such nucleic acid varient is " a silent variant body ", and they are conservative a kind of of varient that modify.Each nucleotide sequence of the coded polypeptide of this paper also refers to the silent variant body that every kind of this nucleic acid is possible.The technician will appreciate that each codon in the nucleic acid can both be modified, thereby produces molecule identical on the function (except AUG and TGG, AUG is unique password of coding methionine(Met), and TGG is unique password of coding colors propylhomoserin).Therefore, the nucleic acid varient of the silence of coded polypeptide all implies in the sequence that each is described.

As for aminoacid sequence, the technician will appreciate that, by nucleic acid, peptide class, polypeptide or protein sequence are carried out single replacement, disappearance or insertion, thereby in encoding sequence, change, add or lack the amino acid of single amino acids or small portion per-cent, this is " the conservative varient of modifying ", and wherein this change has caused amino acid to be replaced by amino acid like the chemofacies.It is well known in the art that intimate amino acid whose conservative replacement table is provided.These conservative varients of modifying are polymorphic variants of the present invention, plant between outside homologue and the allelotrope, and do not get rid of polymorphic variant of the present invention, plant between homologue and allelotrope.

Below 8 groups each the group all contain can guard mutually the replacement amino acid:

1) L-Ala (A), glycine (G)

2) aspartic acid (D), L-glutamic acid (E)

3) l-asparagine (N), glutamine (Q)

4) arginine (R), Methionin (K)

5) Isoleucine (I), leucine (L), methionine(Met) (M), Xie Ansuan (V)

6) phenylalanine (F), tyrosine (Y), tryptophane (W)

7) Serine (S), Threonine (T); With

8) halfcystine (C), methionine(Met) (M)

(referring to, Creighton for example, Proteins, 1984)

For such as macromolecular structures such as polypeptide structures, can be described according to the different structure level.Summary for this structure, can consult for example people such as Alberts, Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I:The Conformation of BiologicalMacromolecules (1980))." primary structure " refers to the aminoacid sequence of a particular peptide." secondary structure " refers to partial orderly three-dimensional structure in the polypeptide.These structures are commonly referred to as (structure) territory.Structural domain is the part of polypeptide, and it forms polypeptide compact unit and long usually 50 to 350 amino acid.Typical structural domain is made of littler structure unit (as beta sheet and alpha-helix)." tertiary structure " refers to the complete three-dimensional structure of polypeptide monomer." quaternary structure " refers to the three-dimensional structure that tertiary structure unit independently forms by non covalent bond.The anisotropy term also is considered to energy terms.

" antibody " refers to a polypeptide, and it has from immunoglobulin gene or its segmental skeleton zone, and can specificity combination and identification antigen.Immunoglobulin gene with recognition function comprises κ, λ, α, γ, δ (σ), ε, μ constant region gene and thousands of immune globulin variable region gene.Light chain is divided into κ or σ.Heavy chain is divided into γ, μ, α, σ or ε, and this defines the kind of immunoglobulin (Ig) successively, IgG, IgM, IgA, IgD and IgE.

The structural unit of a typical immunoglobulin (Ig) (antibody) should comprise the tetramer.Each tetramer is made up of two pairs of identical polypeptide chains, and each is to containing a light chain (approximately 25KD) and a heavy chain (approximately 50-70KD).The N of every chain end has defined about 100-110 or the variable region formed of amino acids more, and identification antigen mainly is responsible in this variable region.Variable light chain (the V of term _L) and variable heavy chain (V _H) refer to these light chains and heavy chain respectively.

Antibody exists with complete immunoglobulin (Ig) form or exists with a large amount of pieces that fully characterize, and these fragments are produced by different peptide enzymic digestion immunoglobulin (Ig)s.Therefore, for example digest antibody below the disulfide linkage of stomach en-in hinge area, thereby produce F (ab) ' ₂, (Fab itself is connected in V by disulfide linkage to the dipolymer of a F (ab) _H-C _HThe light chain in 1 district).By under soft condition, interrupting the hinge area disulfide linkage, can make F (ab) ' ₂Reduction is thus with F (ab) ' ₂Dipolymer is transformed into Fab ' monomer.Fab ' monomer is that Fab with part hinge area is (referring to Fundamental Immunology (Paul compiles, the 3rd edition, 1993) in essence.Although various antibody fragments are to be named by the mode of enzymic digestion according to complete antibody, the technician will appreciate that, these fragments can be with chemical method or DNA recombination method de novo synthesis.Therefore, as used herein, term antibody also should comprise the antibody fragment that complete antibody produces by modifying, and perhaps uses antibody fragment (for example, the strand F of DNA recombinant technology de novo synthesis _v) or the antibody fragment that uses phage to show the library to identify (referring to, people such as McCafferty for example, Nature 348:552-554,1990)

For mono-clonal or Polyclonal Antibody Preparation, arbitrary known technology can both be used (referring to, Kohler ﹠amp for example in this area; Milstein, Nature 256:495-497 (1975); People such as Kozbor, Immunology Today 4:72 (1983); People such as Cole, pp.77-96, Monoclonal Antibodies and Cancer Therapy (1985)).The technology of manufacture order chain antibody (United States Patent (USP) 4946778) can be used to produce polypeptide antibody of the present invention.Transgenic mice or other biological body (as other Mammalss) also can be used to express humanized antibody.Equally, phage display technology can be used to identify specificity be incorporated into the Fab fragment of selected antigenic antibody and different aggressiveness (referring to, for example people such as McCafferty is the same; People such as Marks, Biotechnology 10:779-783,1992).

" chimeric antibody " is a kind of antibody molecule, in this antibody molecule, (a) part in constant region or the constant region is changed, replaces or exchanges, so that antigen binding site (variable region) is connected to different classes of or has changed antibody constant region, effector functional group or the kind of classification or given the diverse molecule of chimeric antibody new property, for example enzyme, toxin, hormone, somatomedin, medicine etc.; Perhaps (b) variable region or wherein a part be changed, replace or exchange with variable region with different or antigen-specific of having changed.

" anti-osteopontin antibody " is a kind of antibody or antibody fragment, and its specific combination is in osteopontin gene, cDNA or its subsequence encoded polypeptide.Anti-EpCAM antibody is defined in a similar manner.

As used herein, " acceptor " comprises and can specificity be incorporated into arbitrary molecule of specific protein (as OPN or EpCAM), and therefore comprise albumen, nucleic acid, carbohydrate or any other molecule.

Term " immunoassay " is to use antibody that antigen is carried out a kind of analysis of specificity bonded.The feature of immunoassay is that the specific combination of utilizing specific antibodies is separated, target and/or quantitative analysis antigen.

When finger protein or peptide, term " specificity (or selectivity) is incorporated into " antibody or " specificity (or selectivity) immune response in " antibody refer to association reaction, and this association reaction is to measure certain albumen whether to be present in determinative in heterologous protein group or the other biological body.Therefore, under specified immunoassay condition, specific antibody is incorporated into the twice that specific protein is background at least, and can not be incorporated into other albumen in the sample basically in a large number.The specific combination of antibody under this condition can need the antibody that has specificity to select because of to specific protein.For example, the anti-OPN polyclonal antibody that obtains from these specific species of rat, mouse or people, thus can selectedly obtain the polyclonal antibody that those with the OPN specific immune response and not do not react with other albumen (except polymorphic variant and the allelotrope of OPN).This screening can have the antibody of cross reaction to be accomplished by the OPN molecule of reducing with other kinds.Various immunoassay mode can be used for screening the antibody that carries out specific reaction with specific protein.For example, solid phase ELISA immunoassay is used to screen the antibody that carries out specific reaction with albumen by routine, (for being used for determining the immunoassay mode of specific immune response and the description of condition, can be referring to, Harlow ﹠amp for example; Lane, Antibodies, A LaboratoryManual, 1988).Typically, specificity or selective reaction double background signal or noise at least, and more typically are to exceed background 10-100 doubly.

Term " difference existence " refers to: compare with the HCC sample of non-transfer or low danger patient's HCC hepatic tissue sample respectively, the biomarker in the HCC tumour of taking from transfer or high-risk patient's HCC hepatic tissue sample is variant on quantity and/or frequency.For example, marker can be polypeptide or nucleic acid, compares with the HCC sample of non-transfer or low danger patient's HCC hepatic tissue sample, and these markers can occur in the HCC tumour of taking from transfer or high-risk patient's HCC hepatic tissue sample high-level or low-levelly.Perhaps, marker is a polypeptide, compares with the HCC sample of non-transfer or low danger patient's HCC hepatic tissue sample, and this polypeptide is detected by frequency in the HCC tumour of taking from transfer or high-risk patient's HCC hepatic tissue sample higher or lowerly.The difference existence of marker can be quantity, frequency or have both at the same time.

If the quantity of polypeptide significantly is different from statistically with the quantity in another sample in a sample, this polypeptide or nucleic acid are exactly that difference exists in two samples so.For example, if appearing in certain sample, polypeptide exceeds at least 120%, at least 130%, at least 150%, at least 180%, at least 200%, at least 300%, at least 500%, at least 700%, at least 900% or at least 1000% than another sample, if perhaps polypeptide be detected in sample therein and in another sample, detect less than, this polypeptide is present in two samples with regard to difference so.

As an alternative or as additional, if the frequency that polypeptide is detected in HCC tumour that shifts or high-risk patient's HCC hepatic tissue sample, detected polypeptide frequency in HCC sample that is higher or lower than in non-transfer to significance or low danger patient's HCC the hepatic tissue sample statistically, this polypeptide is present in two groups of samples with regard to difference so.For example, if observed polypeptide detects other group samples of frequency ratio and is higher or lower than at least 120%, at least 130%, at least 150%, at least 180%, at least 200% at least 300%, at least 500%, at least 700%, at least 900% or at least 1000% in certain group sample, if perhaps polypeptide can be therein be detected in sample and in another sample, detect less than, polypeptide is present in two samples with regard to difference so.

" diagnosis " meaning is exactly: whether or essential attribute the existence of determining pathological symptom or pathological symptom susceptibility is shifted as HCC or HCC.Aspect susceptibility and specificity, diagnostic method can be different.The susceptibility of diagnositc analysis is exactly patient's percentage (per-cent of true positives) of test positive.The patient who is not detected is called false negative.Not having ill and detect negative person in analysis is true negative.The specificity of diagnositc analysis is exactly 1 to deduct false positive rate, and wherein false positive rate is exactly that the people who does not fall ill is detected the male ratio.Though it is a particular diagnostic method may not provide the diagnostic result of determining of disease,, so just enough if this method provides the positive that helps diagnose indication.

The measured quantity of marker is exactly that the digit synbol thing is distributed in quantity in the detected sample.Measured quantity or be to represent (as ug/ml) with absolute quantity, or represent (for example relative intensity of signal) with relative value.

The diagnosis amount of marker is exactly the marker quantity in people's sample, and it is consistent with the diagnosis of the HCC patient's of HCC tumour that shifts or high-risk tissue samples.The diagnosis amount can be to represent (as ug/ml) with absolute quantity, also can represent (for example relative intensity of signal) with relative value.

The contrast amount of marker can be arbitrary quantity or in the quantity of certain scope, and this quantity is used to the measured quantity of isolabeling thing and compares.For example the contrast amount of marker is exactly the marker quantity that occurs in people who does not shift the HCC tumour or low dangerous HCC patient tissue sample.The contrast amount can be to represent (as ug/ml) with absolute quantity, also can represent (for example relative intensity of signal) with relative value.

The spectrophotometer detector refers to a kind of equipment, and it can inject the gaseous ion spectrophotometer in detachable mode, comprises a matrix, and this matrix has the surface that can place the marker that is used to measure.The spectrophotometer detector can contain single matrix or a plurality of matrix.Title has ProteinChip ^, ProteinChip ^Array or chip also refer to the spectrophotometer detector of particular types at this paper.

" matrix " or " detector matrix " refer to provide on its surface sorbent material (as by adhere to, deposition etc.) solid phase carrier.

" sorbent material " refers to can be used to adsorb any material of marker.Here the sorbent material term of Shi Yonging had both referred to the one matter (single entry sorbent material) (for example a compound or a functional group) that contacts with marker, referred to the multiple different material (compound sorbent material) with the marker contact again.Sorbent material in the compound sorbent material is called as " sorbent material kind ".For example, addressable position can comprise compound sorbent material on detector matrix, it is characterized in that having many sorbent material kinds different, that different binding characteristics is arranged (as anionresin material, metal-chelate mixture or antibody).Substrate material itself also can be used to adsorb the part that marker also can be considered to sorbent material.

" absorption " or " reservation " referred to before or after carrying out wash-out with eluent (selectivity threshold value conditioning agent) or washing soln, the detectable combination between sorbent material and marker.

" eluent " or " washing soln " refers to and can be used for the reagent that the aignment mark thing adsorbs sorbent material.Eluent and washing soln all are called as selectivity threshold value conditioning agent.Eluent and washing soln can both be used for wash-out and get rid of the detector stromal surface do not have combined material.

" resolution of marker ", " discrimination " or " parsing " refer in certain sample to have at least a marker to be detected.The meaning of differentiating comprises: through a plurality of markers of separation detection, and difference subsequently detects in certain sample.Resolving does not need one or more markers are separated fully with other biological molecule in the mixture.On the contrary, just enough as long as separation is opened at least one marker and other biological molecular difference.

" gaseous ion spectrophotometer " refers to that measuring parameter can be converted into the instrument of ionic species specific charge when sample is evaporated with ionization.Usually, electric charge of ion band, and mass-to-charge ratio is generally known as quality.For example, the gaseous ion spectrophotometer comprises mass spectrograph, ion migration spectrophotometer and full ionic current survey meter.

" mass spectrograph " refers to the gaseous ion spectrophotometer, and it comprises a sampling system, an ionizer, an ion optics, a mass spectrometer and a detector.

" laser desorption mass spectrograph " refers to utilize with laser is the mass spectrograph of means desorb, evaporation and ionization assay.

" detection " refer to determine detected material existence, do not exist or quantity.

" can detect composition " or " marker " refers to pass through spectrophotometric spectra, photochemistry, biological chemistry, immunochemistry, the detected material of chemical means.For example, useful marker comprises ³²P, ³⁵S, fluorescence dye, electron dense reagent, enzyme (for example being commonly used in the enzyme among the ELISA), vitamin H-streptavidin, digoxin, haptens and protein (existing at their antiserum(antisera) or monoclonal antibody) or the nucleic acid molecule of complementary sequence is arranged with target as horseradish peroxidase.Can detect composition and generally can produce measurable signal, as radioactivity, color development or fluorescent signal, this signal can be used for quantitative sample bonded can detect composition.Signal quantitatively can obtain by for example liquid scintillation counting(LSC), density measurement or fluidic cell surveying.

As used in this application book, " activity " refers to that molecule is as the biological function by the albumen (as osteopontin or EpCAM) of certain genes encoding.This speech comprises biological function, as enzymic activity, with the specific effect of other molecule, on cell or molecular level to adjusting effect of biological activity or the like.

As used herein, term " inhibition " or " restraining effect " refer to relevant target spot molecular function or active down regulation so that function or activity (as enzymic activity or with the specific action of other molecules) detectable decline takes place, or effectively forfeiture.

As used herein, term " antagonist " refers to and can bear the compound of regulating to the biological activity of target molecule (as osteopontin or EpCAM).Antagonist can be finished negative the adjusting by different modes, as transcribe or translation skill by suppressing target gene expression, or disturb the specificity interaction of target molecule and other molecules.

As employed in the context of describing polynucleotide, term " antisense " refers to that the nucleotide sequence of single-chain nucleic acid is complementary at least a portion of the target nucleic acid of the relevant albumen of coding (as osteopontin or EpCAM), promptly with the complementation of " justice " sequence.Complementarity between two strand polynucleotide is based on " A-T G-C " basepairing rule.As the same sequence of sequence " 5 '-AGAT-3 ' " " 5 '-ATCT-3 ' " complementation.Complementarity between target sequence and its antisense polynucleotides typically is 100%, and promptly all bases of antisense polynucleotides are complementary with the target nucleotide base, but different complementary degree also can be arranged, and promptly some mismatched bases can be arranged.Target nucleic acid has remarkable influence with complementary degree between its antisense polynucleotides to the efficient and the intensity of hybridizing.Antisense polynucleotides sequence in this application can be corresponding to the coding region (being exon) or the non-coding region of target nucleic acid.

The concise and to the point description of chart

Fig. 1. according to genetic expression the hepatocellular carcinoma that shifts or do not have to shift is classified.A) by monitoring classification comparative analysis to all 5 clinical group (being P, P-M, PT, PT-M, PN), obtain 143 significance genes (P＜0.0005), utilize 50 primary of these gene pairss and transfer HCC sample to carry out the multidimensional scale analysis.Axle is represented first three main ingredient in these genes.P is the primary HCC of liver internal diffusion; P-M, the transfer damage of P; PT has the primary HCC of TT at portal vein; PN does not have the primary HCC sample that shifts.B) with coming from 383 significance genes (P＜0.0005) that monitoring classification comparison obtains, to hierarchical clustering analysis from 30 primary HCC samples of P, PT and PN group.

Fig. 2. utilize the branch prediction model of the compound co-variation prediction of the cross validation that comes from " omitting single factor (leave-one-out) " classification, to predicting the outcome of shifting and survive and carry out.A) be used for branch prediction model 40 training and HCC patient test.Prediction is based on training set (circle), and it comprises before that in compound co-variation prediction classification the primary HCC sample of used 10 PN and 10 PT and 20 do not have used primary double blinding HCC sample in training program.153 distinguishing significance genes in these two groups have been used in this prediction.B) with 153 the significance gene is arranged, 40 routine primary HCC samples are carried out the multidimensional scale analysis by prediction.Marked patient's identity (ID).C) 40 routine PN, PT and P patient's Kaplan-Meier survival curve.Cross symbols is represented the testing time.

Fig. 3. with shifting the relevant candidate gene of HCC.A) main 30 candidate gene hierarchical clusterings, these expression of gene major part in PT and PT-M group changes, but in the PN group seldom.Each displacement list gene, the single tumor sample of each row representative.In all tumor samples, according to the abundance of a certain gene ratio to all gene abundance intermediate values, each gene just sorts by center association and complete linkage (complete linkage).Pseudo-color hint is differentially expressed: green square, and expression is lower than the transcriptional level of intermediate value; Black squares, expression equals the transcriptional level of intermediate value; Red square, expression is higher than the transcriptional level of intermediate value; Gray squares, No data.Dendrogram is based upon on 10 primary PN (green) and 10 primary PT (redness) sample.B) in being attended by 10 primary PN samples of transfer (black rod) (green rod) and 10 primary PT samples (red rod), the OPN relative expression who obtains by the cDNA microarray analysis leads.C) and D) be in being with or without the primary HCC sample of transfer, the sxemiquantitative RT-PCR analytical results of OPNmRNA level.

Fig. 4. the immunohistochemical analysis of osteopontin in normal liver tissue and the hepatocellular carcinoma.Primary tumor cell (S30 tumour cell) has shown the immune response of tenuigenin osteopontin, and especially in the high dense area of vascular system (figure b and d), but immune response (figure a and c do not appear in (figure b and d) or normal hepatic parenchymal cells in the fibre diaphragm district; Normal hepatocytes 914).50 times of (H﹠amp of enlargement ratio; E, x50).

Fig. 5. the effect of osteopontin in promoting the HCC transfer.A) do the Western trace with the monoclonal anti OPN antibody of rat, determined the concentration of osteopontin in CCL 13, SK-Hep-1 and the Hep3B cell.Mono-clonal beta-actin antibody is used as internal reference.Penetron is used for the quantitative of OPN, and Actin muscle carries out normalized relatively.The OPN water-glass is shown relative multiple.B) be with or without the mouse osteopontin of reorganization, in perhaps being with or without and under the situation in the antibody of anti-osteopontin, hatch CCL 13, SK-Hep-1 and Hep3B cell, and invade chamber (Cell Invasion Chamber) by Matrigel basement membrane cell and measure its intrusion situation.Under each condition, numerical value is the mean value of three observed values, and is expressed as for the diffusion of passing control film (contrast chamber), invades the average percent (adding a standard deviation) of Matrigel matrix and film (matrigel chamber).C) five extra HCC clones (SMMC7721, MHCC97, HuH1, HuH4 and HuH7) react by matrigel matrix and osteopontin neutralizing antibody, and the intrusion situation in this reaction is as above measured.D) subcutaneous injection HCCLM3 cell adds anti-OPN neutralizing antibody (figure below) or does not add anti-OPN neutralizing antibody (last figure), from after the representative (H﹠amp of lung tissue section that obtains of 35 days mouse; E dyeing is amplified 100 times).Arrow is represented the tumour cell grade.E) the different all numbers after giving mouse bare subcutaneous injection HCCLM3 cell, the size of monitoring primary tumor.Data are averages of 10 mouse.F) at mouse bare subcutaneous injection HCCLM3 cell, and the injection or do not inject anti-OPN neutralizing antibody, after 35 days, in nude mice, detect metastasis and form in lung.Based on the grade that shifts, the number of quantitative metastasis.Numerical value is the average of every group of 10 mouse.Group with significance p value (＜0.05) is represented with asterisk.

Fig. 6 .EpCAM is the potential carcinogenesis in the HCC development.A) and b) analyze the expression concentration of the EpCAM of acquisition in the hepatic tissue sample of different chronic hepatic diseases by microarray (a) or RT-PCR (b).C) the Western engram analysis of the monoclonal antibody by anti-EpCAM, the expression of EpCAM in coming from normal people's inoblast (NHF-hTERT), normal liver cell (CCL13) and liver cancer cell (SK-Hep-1, Hep3B, Huh1, Huh4, Huh7 and HepG2).The monoclonal antibody of anti-beta-actin is used as internal reference.D) analyze Hep3B, the Huh1 of acquisition and the increment situation of Huh4 cell by MTT, numerical value is the mean value of three independent experiments.E), measure siRNA EpCAM is expressed effective restraining effect by the Western engram analysis.F) by the restraining effect of MTT assay determination EpCAM siRNA to the growth of Hep3B cell.

Detailed Description Of The Invention

In the world today, hepatocellular carcinoma (HCC) is one of the most general and the most aggressive malignant tumour, and is very in vogue in the Asia and Africa, and in Europe relative (people such as Parkin, CA Cancer J.Clin.49:33-64, the 1999 less with North America; People such as Pisani, Int.J.Cancer 83:18-29,1999).Recent research shows that in the past twenty years, HCC significantly increases (people such as Taylor-Robinson, Lancet 350:1142-1143,1997 in the incidence of the U.S. and Britain; El-Serag and Mason, N.Eng.J.Med.340:745-750,1999).Most of HCC patients are owing to the prediction that falls behind causes and can't give treatment to.Although by the life-span that can make some patient obtain to prolong to developing HCC patient's routine inspection, but have many patients be diagnosed as late period HCC and by deprived existence (referring to, for example, people such as Yang, J.Cancer Res.Clin.Oncol.123:357-360,1997; People such as Izzo, Ann.Surg.227:513-518,1998).Though the qualified surgical operation interventional therapy of carrying out of sub-fraction HCC patient is little for the raising of long-term survival quality.The extreme HCC prediction that falls behind mainly is because the high relapse rate behind the excision, or because pylic intrusion develops into secondary tumour in the liver, or owing to be diffused into other interior positions of liver, yet the generation of the outer secondary tumour of liver general (referring to, for example, people such as Genda, Hepatology 30:1027-1036,1999).These document description livers are main target organs that HCC shifts.This animal model system and portal vein be shift among the patient that the main route of secondary tumour in the liver takes place the HCC cell and be confirmed (referring to, for example, people such as Mitsunobu, Clin.Exp.Metastasis 14:520-529,1996).The single-minded feature of HCC emphasizes to develop a kind of necessity of accurate molecular simulation model, and purpose is that the patient of secondary tumour provides better diagnosis and treatment target spot in the liver for those are had.

Recent research mainly concentrate on the single candidate gene (referring to, for example, people such as Osada, Hepatology24:1460-1467,1996; People such as Guo, Hepatology 28:1481-1488,1998; People such as Hui, Int.J.Cancer84:604-608,1999).This may not have enough accurately to reflect the biology essence of transfevent HCC.Microarray technology provide the opportunity in full genome, seeking disease related gene and express (referring to, for example, people such as Schena, Science 270:467-470,1995).Aspect the reaction after the development process of tumour, prognosis result or the treatment, this approach has carried out successful molecular classification (people such as Alizadeh, Nature403:503-511,2000 to multiple human malignancies; People such as Bittner, Nature 406:536-540,2000; People such as Perou, Nature406:747-752,2000; People such as Khan, Nat.Med.7:673-679,2001; People such as Pomeroy, Nature 415:436-442,2002; People such as Shipp, Nat.Med.8:68-74,2002).A plurality of reports have related to gene expression profile (people such as Okabe, Cancer Res.61:2129-2137,2001 of primary HCC sample; People such as Xu, Proc.Natl.Acad.Sci.U.S.A.98:15089-15094,2001).Yet, not clear with the molecular signal that transfevent HCC patient prognosis feature is relevant.

Use can be studied the institute that is correlated with transfer and change based on the gene expression atlas of cDNA microarray.Originally purpose is the gene that shifts damage in the liver that can distinguish primary tumor and its coupling in order to identify.Disclose, the transfer damage is a undistinguishable with primary tumor in the liver, and it and tumour size, microencapsulation and patient's age have nothing to do, although the HCC that does not have primary to shift can make a distinction with having primary transfer HCC.Above data explanation, the variation that helps to shift in the liver starts in primary HCC.In addition, a gene that plays an important role is that osteopontin (a kind of secretor type phosphorprotein) appears in the HCC transfer.External, the expression of crossing of osteopontin is correlated with primary HCC, this primary HCC ability of having metastatic potential and invading liver neoplasm derived cell system wherein, and can in and the antibody capable of osteopontin in the intrusion of external effective blocking-up HCC cell and also can block the transfer of HCC cell in the lung in vivo.These researchs are clear and definite, and osteopontin both can be used as to be used for determining whether HCC patient has the molecular marked compound of metastatic potential, also can be used as the potential treatment target spot of treatment transfevent HCC.

Similarly method is used to develop the genetic expression predictive model, develops into the possibility of HCC so that predict those chronic hepatitis patients.Patient's gene expression atlas that the patient's gene expression atlas of high-risk trouble HCC on the epidemiology is suffered from HCC with low danger on the epidemiology is compared, can identify cell marker, thereby can identify the patient of the high-risk strategical vantage point of chronic hepatopathy meeting to the HCC development.Those have serious hepatic diseases patient to comprise that those are diagnosed as hepatitis B, third liver, pigmentary cirrhosis, the inferior disease of Weir, alcoholic liver, autoimmune hepatitis and primary liver and gall hardened people.The disease that high-risk is brought out early-stage cancer has chronic viral hepatitis B, chronic disease liver, pigmentary cirrhosis and hepatolenticular degeneration.The disease that low danger is brought out for early-stage cancer has alcoholic liver, autoimmune hepatitis and the sclerosis of primary liver and gall.The EpCAM gene of finding in patient's body of serious hepatopathy is proved, and it is with to bring out high-risk HCC relevant.By suppressing the expression of EpCAM, the growth-inhibiting phenomenon of liver cancer cell is observed, has determined the vital role that EpCAM is risen in the HCC development thus, and can be used as the treatment target spot that the prevention chronic hepatitis patients develops into HCC.

A concrete aspect of the present invention has just provided a kind of method, and it has transfevent HCC or have the intravital regulatory gene altogether of the patient who develops into HCC potential to carry out cluster analysis doubtful, thereby forms gene expression atlas.This joint provides and has carried out the more detailed argumentation of cluster analysis to being total to regulatory gene.

The I.DNA microarray analysis

A. the classification by the cluster analysis gene expression atlas

For many application of the present invention, be necessary to find the expression map of basi gene in non-transfevent HCC sample, transfevent HCC sample, high-risk developing HCC sample and the developing HCC sample of low danger of common adjusting.The preferred example of determining the gene expression atlas that these are basic relate to clustering algorithm (for the summary of clustering algorithm, can consult Fukunaga, 1990, Statistical Pattern Recognition, 2 editions., Academic Press, San Diego; Everitt, 1974, Cluster Analysis, London:Heinemann Educ.Books; Hartigan, 1975, Clustering Algorithms, New York:Wiley; Sneath and Sokal, 1973, Numerical Taxonomy, Freeman; Anderberg, 1973, Cluster Analysis for Applications, Academic Press:NewYork).

Use in the example of cluster analysis at some, in the biological specimen of different sources, a large amount of expression of gene can be monitored to.The data sheet that contains the genetic expression observed value has been used for cluster analysis.Cluster analysis meeting computing on the data sheet of m * k dimension, wherein m refers to the sum of conditioned disjunction fluctuating factor, k is the gene dosage of having measured.

There are many clustering algorithms to can be used for cluster analysis.When needs formed bunch, clustering algorithm can use dissimilarity or the distance between object.In some instances, the distance of using in the hyperspace refers to Euclidean distance (Euclideandistance).Euclidean distance can by square, thereby to separating on the farther object weight that increases gradually is set.Perhaps, the standard of measurement of distance can be a manhatton distance.In other examples, data sheet is not supervised the hierarchical clustering analysis, can use CLUSTER or TREEVIEW software (people such as Eisen, Proc.Natl.Acad.Sci.USA, 95:14863-14868,1998) to carry out, these softwares are to have utilized association of intermediate value center and complete linkage.

Various bunch of chain (linkage) rule can be used for method of the present invention.List is chain to be a kind of nearest neighbor method, and it measures the distance between two hithermost objects.On the contrary, complete linkage method is to determine distance by the ultimate range between wantonly two objects in different bunches.This method is specially adapted to the situation that gene or other cellular components constitute natural different " clumping (clump) ".Perhaps, the average of unweighted pairing-group (pair-group) has defined the mean distance between all pairing objects in two differences bunch.Gene or other cellular constituents are being carried out cluster analysis when forming the different clumping of nature, this method is also very useful.At last, the pairing of weighting-group averaging method also can be used.This method is identical with unweighted pairing-group averaging method, and difference is the size of each bunch is used as weight.This method is specially adapted to the situation that bunch size has wide variation.(Sneath and Sokal, 1973, Numerical taxonomy, San Francisco.W.H.Freeman ﹠amp; Co.).The chain rule of other bunches, for example unweighted and pairing-group centre of moment and WardShi algorithm weighting also can be used for examples more of the present invention.Can consult document Ward, 1963, J.Am.Stat Assn.58:236; Hartigan, 1975, Clustering algorithms, NewYork:Wiley.

In a more preferred example, the cluster analysis of using is BRB-ArrayTools software, this is the integrated software package by the biometrics research branch exploitation of American National ICR, be used for cDNA microarray gene expression data is carried out visual and statistical study, can be used for the analysis of no worker monitor and the analysis of monitoring." classification compare tool (Class Comparison Tool) " based on single argument F check is used in the gene of seeking differential expression between the predetermined clinical group that the significant difference level is P＜0.001 or 0.002.Based on 2000 arrangements at random, the arranged distribution state of F statistical study also can be used for determining significant difference.By using of the arrangement of 2000 P values at random less than 0.001 significant difference level, simultaneously according to gene expression atlas, can use the compound co-variation forecasting tool of multivariate (Compound Covariate Predictor with the test of " omitting single factor (leave-one-out) " cross validation, CCP), predetermined clinical group is classified.In the step of each cross validation, a sample is omitted, and creates a multivariate CCP based on gene, and wherein said gene is in the training group of being made up of the sample that is not omitted, remarkable monotropic gene under specified level.CCP is used to the sample classification after omitting, and indicates classification then and is correctly or mistake.All to carry out repetition for all samples after one of each eliminating.The mis-classification ratio of total cross validation is determined like this.The mis-classification ratio significance statistically of cross validation determines that by the complete cross validation program that data is repeated 2000 times the member that wherein classifies is a random permutation.CCP is based upon on the weighted linear combination basis of genetic expression variable, wherein said variable is significantly monotropic in the training group, and its weight is added up corresponding to t-, as people such as Radmacher, Journal of Computational Biology (in the publication) is described in 2002.The example of clustering tree output is presented among Fig. 1 and Fig. 3 (also can consult embodiment 1 hereinafter).

Gene expression profile can define based on many littler branches in the tree, perhaps defines with many bigger branches by cut down clustering tree on different levels.The felling level must be complementary with the clinical group number of desired difference.If only there is seldom or do not have information formerly for the quantity of group, this clustering tree should be divided into true different many branches so.Minimum distance value defines between " true different " available single branch.This distance is the ordinate zou (consulting Figure 1B) that connects two horizontal lines of ramose.Representative value wherein 0 is meant complete association in the 0.2-0.4 scope, the 1st, the nulling association, but in the training set the less or test of good data more after a little while, representative value can be bigger, perhaps when the training intensive data better and test more for a long time, representative value can be littler.

More preferably, the objective examination of " true different " available statistical significance to each bifurcated in the clustering tree defines.In one aspect of the invention, by on predetermined significance level, use 2000 random permutations, and, define the objective examination with compound co-variation forecasting tool with the test of " omitting single factor " cross validation.Traction improvement (tractional improvement) distribution that obtains with the CCP program is the assessed value to distributing under null hypothesis theory (being that specific classification is a right or wrong).

Clustering method in the present invention be on the other hand, the definition with underlying carrier is provided, be used for total hereinafter described collection of illustrative plates planning.

B. Pu comparison and classification

The method of finding medicine that provides is provided.In an example, gene expression profile defines with cluster analysis.Gene in the gene expression profile is revealed under condition interested to be potential adjusting altogether.Can further study common regulatory gene and whether relate to the adjusting approach.Identify the gene that relates to the adjusting approach, can be design and screening new drug provides useful information.

In some example of the present invention, the screening drug candidate is used for the treatment of.In an example, desired pharmaceutical activity can influence certain specific heredity and regulate approach.In an example,, screen drug candidate according to the ability of influence corresponding to the gene expression profile of the approach of adjusting.In another example, the expectation new drug replaces existing medicine.In an example, the design spectrum of drug candidate is compared with existing medicine, has with the existing similar activity of medicine so that determine which drug candidate.

In some example, method of the present invention is used to explain tree derivation and kinetics.When acceptor was excited (or blocking-up) by part, the excitability of downstream passages may be different, and this depends on the molecular structure territory of accurate transient expression spectrum and part and acceptor interaction.It is phenotypic difference that different ligands causes the simple case of different effect, this difference results from the response to agonist, partial agonist, anti-antagonist and antagonist, and expects that this difference can result from the activated response of covalent linkage to differing molecular zone on the combination of non covalent bond and the acceptor.Consult Ross, (people such as Gilman edits Pharmacodynamics:Mechanisms of Drug Action and the Relationship between DrugConcentration and Effect in The Pharmacological Basis of Therapeutics, McGraw Hill, New York, 1996).Fig. 4 A has stated two kinds of possible differential responses in the path cascade.

Among some embodiment of the present invention, can study with method of design of the present invention, so that observed instantaneous reaction is reduced to the receptor/ligand effect that response gene is made as the acceptor that with OPN is part.Especially in some particularly preferred examples, gene expression related spectrum and instantaneous spectrum have been found.The instantaneous reaction spectrum of a large amount of genes is arrived predetermined gene expression profile by projection (projected), thereby obtains the planning spectrum of instantaneous reaction.This planning process has been simplified observed reaction, and therefore different instantaneous reactions can be detected more accurately and distinguish.

C. the explanation of diagnostic use

An aspect of of the present present invention provides the method for the disease of diagnosing human, animal and plant.This method can be used for the process of monitoring of diseases development and the validity of treatment equally.

In one embodiment of the invention, can carry out a large amount of gene expression analysis to patient's cell sample (Tathagata is from the biopsy sample of transfevent HCC patient's illing tissue).According to the definition of gene expression profile, this gene expression profile is formulated for the expression values spectrum of genetic expression.The spectrum that planning is good is compared with the contrasting data storehouse that contains contrast planning spectrum.If in database, patient's planning spectrum is best with cancer collection of illustrative plates coupling, and patient's pathological tissue is diagnosed as cancer so.Similarly, when optimum matching is the collection of illustrative plates of other diseases, that just is diagnosed as this kind disease.

In another embodiment, tissue samples obtains from patient's tumor tissues.This tissue samples is carried out a large amount of related gene expression analyses.According to the definition of gene expression profile, this gene expression profile is formulated for the expression values spectrum of genetic expression.The spectrum that planning is good is compared with previous planning spectrum from identical tumour, change to determine the expression in the gene expression profile.Determine with the contrast storehouse whether the change of gene expression profile is indicating tumor development (as shifting).Similarly method can be used for determining other diseases or disorderly stage.In the treatment in patient's collection of illustrative plates the variation of gene expression profile expression values can be used for the validity of monitor therapy, for example, by before the treatment relatively and the planning collection of illustrative plates after the treatment.

D. the enforcement of assay kit

In preference, method of the present invention can be accomplished by using the test kit of measuring biological specimen reaction or state.Such test kit contains microarray, for example following described microarray of paragraph (chip).Chip in these test kits comprises solid phase (a for example surface), and probe hybridization in or be incorporated into the known location of solid phase.Preferably, these probes are made up of known different nucleic acid, and each nucleic acid can be with the RNA or the cDNA molecular hybridization that derive from this nucleic acid.Especially, the probe that contains in the test kit of the present invention is can hybridize specifically in the nucleotide sequence that is derived from RNA, and wherein the increase of known this RNA or minimizing are corresponding to the fluctuation by active certain specific protein of this kit measurement.Probe in the test kit of the present invention should be got rid of the nucleic acid of the hybridization of those nothing to do withs RNA basically, and for the fluctuation by active certain specific protein of this kit measurement (as osteopontin), these RNA can not increase.

In preference, test kit of the present invention has the database (as above-mentioned database) of gene expression profile definition simultaneously or allows the telecommunication network computer to use the insertion authority book of above-mentioned database.

On another preference, test kit of the present invention further comprises the planning that is used for expression map and the software of analysis, and this software can be downloaded in the internal memory of computer system, for example above described in the trifle and in embodiment 1, set forth like that.Expression pattern analysis software in the test kit of the present invention is with being equal on the expression pattern analysis software nature described in the top embodiment 1.

Be used to implement other test kits of analytical procedure of the present invention, be conspicuous to those skilled in the art, thereby be included in the appended claim.Particularly, Fu Sui claim be used to comprise be used to carry out the inventive method, conspicuous other program structures to those skilled in the art.

E. measure the method for biological respinse collection of illustrative plates

The present invention has utilized the ability of assaying reaction, and these reactions are that living things system is made at a large amount of different fluctuations.This section provides some exemplary process for measuring biological respinse.One skilled in the art will recognize that the present invention is not limited to the method that following specific mensuration living things system is reacted.

1. utilize the DNA chip to carry out transcription analysis

The present invention is specially adapted to the analysis of gene expression profile.One aspect of the present invention provides the method for determining to be total to the regulatory gene expression map based on the genetic expression cognation.Some embodiments of the invention are based on the measurement to the genetic transcription rate.

Transcription rate can obtain measuring (as described in next joint) by nucleic acid chip or nucleic acid analogue probe hybridization technique, perhaps obtains measuring by other gene expression techniques, for example in the technology described in the joint subsequently.Yet, in case determined, result or be the absolute magnitude or the relative quantity of transcript, or be reply data, and comprise the numerical value of expression RNA abundance rate, wherein RNA abundance rate is through being commonly used to reflect DNA expression rate (when not having the difference of RNA degradation rate).

In various different embodiment of the present invention, also can measure biological aspect aspect such as translation state, active condition or admixture except transcriptional state.

Preferably, the mensuration of transcriptional state can be by obtaining with the DNA chip hybridization, and the DNA chip is narrated in this section.Some additive method of measuring transcriptional state will be in the narration of the back of this trifle.

In preference, the present invention has used the DNA chip.The DNA chip can be used for the transcriptional state in the analyzing biological samples, and is particluarly suitable for being exposed under the gradient drug levels at different levels or under the gradient fluctuation of relevant bio signal approach, measures the transcriptional state of biological specimen.

In one embodiment, the preparation of DNA chip be by with the oligonucleotide hybridization of detectable mark in chip, wherein this oligonucleotide represented the mRNA transcript that is present in the cell (as fluorescently-labeled, from the synthetic cDNA that gets of the total mRNA of cell).Chip is exactly a surface that has in conjunction with the oldered array in (as hybridization) site, and these sites are used in conjunction with cell or a large amount of genes of organism genome, more preferably major part or the almost product of full gene.Chip can prepare with many modes, and several will the narration below arranged.Yet the chip of preparation has some preferred features: chip has circulation ratio, allows a plurality of copies of certain certain chip of preparation and is easy to mutual comparison.Preferred chip is small-sized, generally all less than 5 ²Cm, and they are used under the association reaction condition (as nucleic acid hybridization) stable material and make.In the chip a certain binding site or unique binding site collect will specific combination individual gene in cell product.Although a plurality of physical bond site (back is referred to as " site ") can be arranged for each specific mRNA, describe for the ease of clear, below argumentation can suppose to have only single site.

Should be understood that when and cell RNA complementary cDNA be synthesized, and when under suitable hybridization conditions, hybridize, corresponding to the hybridization level in the site of arbitrary gene, can reflect the mRNA level of interior this genetic transcription of cell in the chip with microarray (chip).Such as, during when (as using fluorophore) that can detect ground mark, with total cell mRNA complementary cDNA and microarray hybridization, can signal very little or do not have a signal generation (as fluorescent signal) on the array corresponding to the site (promptly can combine) of non transcribed gene in the cell with the gene product specificity; And, then can produce stronger signal for the extensive gene that exists of the mRNA of coding.

In preference, the cDNA of two kinds of different cells and the hybridization of the binding site of microarray.In drug reaction, a kind of biological sample contacts with medicine, and the another kind of biological sample of same type does not contact with medicine.In pathway response, a cellular exposure is in the path disturbance, and another cell of same type is not exposed to the path disturbance.From the cDNA different methods mark of two kinds of cells, the convenient differentiation.In one embodiment, such as, through the cDNA of the cell of a kind of drug treating (or being exposed to the path disturbance), synthetic with fluorescein-labeled dNTP; From the cDNA of another kind, then synthetic with the dNTP of rhodamine marker without the cell of drug treating.During when two kinds of cDNA mixing and with microarray hybridization, can measure the signal relative intensity of every kind of cDNA group in each site on the array, thereby detect the relative mistake of specific mRNA abundance.

In above-mentioned example, when fluorophore was stimulated, the cDNA that drug treating is crossed the cell of (or path turbulent) showed green fluorescence, and the cDNA of untreated cell shows red fluorescence.The result is, when drug treating when the relative abundance of a certain specific mRNA is invalid in the pair cell directly or indirectly, this mRNA is distributed in two kinds of cells together with waiting, in case and reverse transcription, cDNA red-label and Green Marker can exist on an equal basis.When hybridizing, can send two kinds of distinctive wavelength of fluorophore (and presenting brown after the combination) corresponding to the binding site of this RNA in microarray.On the contrary, when the cell that contacts with medicine is with a kind of can directly or indirectly increase the drug treating of mRNA level in the cell time, green fluorescence can increase the ratio of red fluorescence intensity.If medicine reduces the mRNA level, this strength ratio can reduce.

With Two Colour Fluorescence mark and detection method to determine the method for changes in gene expression, people such as for example Shena, " Quantitative monitoring of gene expression patterns with a complementary DNAmicroarray; " Science 270:467-470, description is arranged in 1995, and the document is incorporated herein by reference in full at this.Advantage with the cDNA of two kinds of different fluorophore marks is, can obtain a direct internal contrast comparative figure, and the caused variation of test conditions (as hybridization conditions) fine difference can not influence analysis subsequently corresponding to every kind of array gene mRNA level.It should be understood that, can use the cDNA of individual cells, and comparative example is as the absolute quantity of specific mRNA in drug treating or path turbulent cell and untreated cell.

2. the preparation of microarray

Microarray (chip) is known in the art, and it contains a surface, on this surface with the corresponding probe specificity of gene product (as cDNAs, mRNAs, cRNAs, polypeptide and fragment thereof) sequence hybridize in or be incorporated into known site.In one embodiment, microarray is a kind of array (being matrix), and wherein a kind of discrete binding site of product (as albumen or RNA) of genes encoding is represented in each site.Simultaneously, binding site has wherein been represented most of or most gene product in the organism genome.In a preferred embodiment, " binding site " (hereinafter " site ") is nucleic acid or nucleic acid analog, they can with a certain specific homology cDNA specific hybrid.The nucleic acid of binding site or analogue can be, for example synthetic oligomer, full-length cDNA, than total length short cDNA or gene fragment.

Although in preference, microarray comprises in the organism target gene group binding site of all or nearly all gene product, all be this comprehensive be not essential.Usually, microarray includes in the genome about at least 50%, and generally about at least 75%, more common about at least 85%, more general about at least 90%, the most general about at least 99% gene binding site.Microarray should have the gene binding site relevant with medicine or biological pathway effect.Differentiated to open " gene " of reading frame (ORF) should contain at least 50,75 or 99 amino acid, and its mRNA is in some transit cell records of organism (for example, if unicellular) or multicellular organisms.Number gene in the genome can pass through the mRNA quantity survey of organism expressing, or infers according to the part that genome has fully been studied.When the organism genome of being studied is checked order, the quantity of ORFs can be determined and can determine the mRNA coding region by the analyzing DNA sequence.Such as, the genome of yeast saccharomyces cerevisiae is checked order fully, and reports that containing 6275 approximately is longer than 99 amino acid whose open frames (ORFs) of reading.The analysis of these ORFs is shown that 5885 ORFs have protein product (people such as Goffeau, 1996, Life with 6000 genes, Science274:546-567, the document is incorporated herein by reference in full).Comparatively speaking, human genome is estimated to contain to have an appointment 5 * 10 ⁴Individual gene.

3. prepare microarray nucleic acid

As mentioned above, one with " binding site " of specific homology cDNA specific hybrid normally attached to nucleic acid on this binding site or nucleic acid analog.In one embodiment, the binding site of microarray is the DNA polynucleotide, and it is corresponding at least one fragment of each gene of organism genome.These DNA can obtain genomic dna, cDNA (as passing through RT-PCR) or the amplification of cloned sequence by polymerase chain reaction (PCR) etc.According to the known array of gene or cDNA, select the PCR primer, thereby amplification obtains unique fragment (promptly and other fragment on the array not more than the fragment of the identical sequence of 10 bases).The amplification condition of available computers programdesign Auele Specific Primer and optimization.Referring to as Oligo 5.0 editions (National Biosciences).If the binding site of very long gene, fragment that can the nearly 3 ' end of amplification gene when widow-dT primer cDNA probe and microarray hybridization, is shorter than the effectively combination of probe of total length like this.Typically, each gene fragment length is between 50bp and 2000bp on the microarray, and more typically between 100bp and 1000bp, common length is between 300bp and the 800bp.The method of PCR is known, and is editing 1990 as people such as Innis, PCR Protocols:A Guide to Methods and Applications, Academic Press Inc., San Diego, description is arranged Calif., and the document is incorporated by reference in this text to be examined.Clearly, computer-controlled automatic system can be used for effectively separating and amplification of nucleic acid.

The other method of synthetic microarray nucleic acid is with N-phosphonic acids acyl or phosphonic acid amide chemical process synthetic polyribonucleotides or oligonucleotide (people such as Froehler, 1986, Nucleic Acid, Res 14:5399-5407; People such as McBride, 1983, Tetrahedron Lett.24:245-248).The synthetic sequence length is between 15 and 500 bases, more typically between 20 and 50 bases.In certain embodiments, contain non-natural base in the synthetic nucleic acid, as Trophicardyl.As mentioned above, nucleic acid analog can be used as the binding site of hybridization.The example of a suitable nucleic acid analog is that peptide nucleic acid(PNA) (is seen people such as Egholm, 1993, PNA hybridizes to complementary oligonucleotides obeying theWatson-Crick hydrogen-bonding rules, Nature 365:566-568; Also can be) referring to U.S. Patent No. 5,539,083.

In another embodiment; in conjunction with plasmid or phage clone, cDNA (as expressed sequence tag) or its insertion sequence (people such as Nguyen of (hybridization) site from gene; 1995; Differential gene expression in the murine thymusassayed by quantitative hybridization of arrayed cDNA clones, Genomics 29:207-209).In another embodiment, the polynucleotide of binding site are RNA.

4. the adhesion of nucleic acid and solid phase surface

Nucleic acid or analogue are attached on the solid phase carrier, and solid phase carrier can be to use glass, plastics (as polypropylene, nylon), and polyacrylamide, nitrocellulose or other material are made.Is to xerox on the sheet glass nucleic acid adhesive to a kind of preferable methods on surface, as people such as Schena, 1995, described in the Quantitative monitoring ofgene expressionpatterns with a complementary DNA microarray, Science 270:467-470 like that.This method is particularly useful for making the microarray of cDNA.Referring to people such as DeRisi, 1996, Use of a cDNAmicroarray to analyze gene expression patterns in human cancer, Nature Genetics 14:457-460; People such as Shalon, 1996, A DNA microarray system for analyzing complex DNA samplesusing two-color fluorescent probe hybridization, Genome Res.6:639-645; And people such as Schena, 1995, Parallel human genome analysis; Microarray-based expression of 1000 genes, Proc.Natl.Acad.Sci.USA 93:10539-11286.

Second kind of preferred method for preparing microarray is the highdensity oligonucleotide array of preparation.Being created in the technology that contains the array of thousands of kinds and definite sequence complementary oligonucleotide on the qualification site knows, available photolithography technology carries out that surface in situ is synthetic (sees people such as Fodor, 1991, Light-directed spatiallyaddressable parallel chemical synthesis, Science 251:767-773; People such as Pease, 1994, Light-directed oligonucleotide arrays for rapid dna sequence dna analysis, Proc.Natl.Acad.Sci.USA91:5022-5026; People such as Lockhart, 1996, Expression monitoring by hybridization to high-density oligonucleotide arrays, Nature Biotech 14:1675; United States Patent(USP) Nos. 5,578,832; 5,556,752; With 5,510,270, every piece of document all is incorporated by reference in this text to be examined), method of also available other fast synthetic and oligonucleotide that deposition limits (people such as Blanchard, 1996, High-Density, Oligonucleotide arrays, Biosensors ﹠amp; Bioelectronics 11:687-90).When these methods of application, the oligonucleotide of known array (as the 20-polymers) is gone up directly synthetic on surface (as the slide of deriving).Usually, the array of generation comprises the multiple probe at every kind of target transcripton.Oligonucleotide probe can be used for detecting montage mRNAs or as various dissimilar contrasts.

The preferred approach of another kind of preparation microarray is by using ink jet printing process directly synthetic oligonucleotide on solid phase.

Also can adopt other method for preparing microarray, as mask (Maskos and Southern, 1992, Nuc.Acids Res.20:1679-1684).In theory, the array of any kind, (see Sambrook andRussell as the dot blot on the nylon Hybond membrane, Molecular Cloning:A Laboratory Manual 3 editions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 2001) can adopt.Yet as the art technology people be familiar with, very little chip is preferred, because the hybridization volume is littler.

5. the probe of complex sign

The method for preparing total RNA and poly-(A)+RNA is known, and in people such as Sambrook (the same) description is arranged.In one embodiment, extracting RNA from the interested multiple biological sample of the present invention, wherein with after the guanidine thiocyanate cracking with CsCl centrifugal (people such as Chirgwin, 1979, Biochemistry 18:5294-5299).Or, can use TRIzol reagent (Life Technologies), from sample, extract total RNA according to operational manual.Poly (A)+RNA is with widow-dT cellulose selective (seeing Sambrook and Russell, the same).Useful biological sample comprises the liver sample of normal liver sample, non-canceration and from the sample of the clinical sample of making a definite diagnosis.

Available few dT-primer or random primer reverse transcription mRNA prepare the cDNA of mark, and the method for these two kinds of reverse transcriptions all is (referring to, as Klug and Berger, 1987, Methods Enzymol.152:316-325) known.Reverse transcription can carry out when having dNTP to exist, and used dNTP is connected with detectable marker, preferably fluorescently-labeled dNTP.Or, strand mRNA can be under the condition that the dNTPs of mark exists through the sense-rna of double-stranded cDNA in-vitro transcription complex sign (people such as Lockhart, 1996, Expression monitoring by hybridization to high-density oligonucleotide arrays, Nature Biotech.14:1675, the document is incorporated herein by reference in full).In another embodiment, cDNA or rna probe can not have in the presence of the detectable synthetic, and then mark, as passing through in conjunction with biotin labeled dNTPs or rNTP, or with some similar methods (carrying out photo-crosslinking), add streptavidin (as the coupled streptavidin that phycoerythrin is arranged) or its Equivalent of mark then as psoralene derivative and RNA with vitamin H.

If use fluorescently-labeled probe, it is known that many suitable fluorophores are arranged, comprise fluorescein, lissamine rhodamine, phycoerythrin, rhodamine (Perkin ElmerCetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) etc. (referring to, as Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press SanDiego, Calif.).Should be understood that the fluorophore that to select different emission spectrums, so that distinguish.

In another embodiment, the marker outside the use fluorescent marker.Such as, can use radioactively labelled substance or have a pair of radioactively labelled substance of different emission spectrums (to see people such as Zhao, 1995, High density cDNA filteranalysis:a novel approach for large-scale, quantitative analysis of gene expression, Gene156:207; People such as Pietu, 1996, Novel gene transcripts preferentially expressed in humanmuscles revealed by quantitative hybridization of a high density cDNA array, Genome Res.6:492).But because radioactive particulate has scattering thereby needs bigger at interval binding site, therefore using isotopic label is time good embodiment.

In one embodiment, the cDNA of mark is containing 0.5mMdGTP, dATP, dCTP, 0.1mM dTTP, the fluorescence deoxynucleotide (as, 0.1mM rhodamine 110 UTP (Perken Elmer Cetus) or 0.1mM Cy3 dUTP (Amersham)) and reversed transcriptive enzyme (as SuperScriptTM II, LTIInc.) in the mixture, synthesized in 42 ℃ of incubations 60 minutes.

6. with the hybridization of microarray

Select the nucleic acid hybridization and the wash conditions of optimization, make probe " specificity combination " or " specific hybrid " in special array site, be probe hybridization, connect or be incorporated on the sequence array site of complementary nucleic acid sequence, and do not hybridize in the site of incomplementarity nucleotide sequence.As used herein, if, under the standard basepairing rule, do not have mispairing when short that in two polynucleotide during smaller or equal to 25 bases, if or during greater than 25 bases, mispairing just can not surpass 5%, and to be considered to another be complementary to a polynucleotide sequence so.Preferably, oligonucleotide complementation (not having mispairing) fully.Clearly, contain the hybridization analysis of negative control by use, can make specific hybridization conditions produce specific hybrid (referring to, the same as people such as Shalon, and people such as Chee, the same).

The hybridization conditions of optimizing depends on the length (polynucleotide that length surpassed 200 bases as oligomer) and the type (as RNA, DNA, PNA) of label probe and immobilization polynucleotide or oligonucleotide.General parameter for special (being rigorous) hybridization conditions of nucleic acid, people such as Sambrook, the same, with people such as Ausubel, 1987, CurrentProtocols in Molecular Biology, Greene Publishing and Wiley-Interscience has description among the New York.If people's such as employing Schena cDNA microarray, typical hybridization conditions is to add 0.2%SDS at 5xSSC, hybridized 4 hours down for 65 ℃, then in 25 ℃ of washings (1xSSC adds 0.2%SDS) in low rigorous lavation buffer solution, then in 25 ℃ of washing 10 minutes (0.1xSSC adds 0.2%SDS) (people such as Shena in the rigorous lavation buffer solution of height, 1996, Proc.Natl.Acad.Sci.USA, 93:10614).The available hybridization conditions also can be referring to as Tijessen, 1993, Hybridization With Nucleic Acid Probes, Elsevier Science Publishers B.V.andKricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press San Diego, Calif.

7. signal detection and data analysis

If use fluorescently-labeled probe, the fluorescent emission on each site of transcript array all can be observed at the confocal laser microscopically.The most handy Axon GenePix 4000 scanners of fluorescence intensity are measured.In one embodiment, use suitable exciting light that two fluorophores are respectively once independently scanned.Perhaps, can use one laser under two kinds of fluorophore certain wavelengths, to make sample simultaneously luminous, and the emission of analyzing two fluorophores simultaneously (is seen people such as Shalon, 1996, A DNA microarray system for analyzing complex DNA samples usingtwo-color fluorescent probe hybridization, Genome Research 6:639-645, the document is incorporated herein by reference in full).In a preferred embodiment, array uses a lasing fluorescence scanning imaging instrument that has a computer-controlled X-Y coordinate and a micro objective to scan.Two fluorophores detect by wavelength separated and by two photomultiplier with multi-thread mixed gas laser continuous agitation, the light of emission.The fluorescence laser scanning device is people such as Schena, and 1996, Genome Res.6:639-645 and institute thereof draw description in the reference.Perhaps, people such as Ferguson, 1996, the fiber optics bundle of describing among the NatureBiotech.14:1681-1684 also can be used for mRNA abundance level is detected in a large amount of sites simultaneously.

Signal is recorded, and in preference by Computer Analysis, as use 12 bit simulators to digiboard.In one embodiment, the image of scanning carries out deblurring with image program (as Hijaak Graphics Suite), paints the average hybridization data table that each each wavelength of site is set up in the lattice programanalysis with image then.If necessary, can measure the correction of " crosstalk " (or overlapping) of setting up between two fluorescence paths by experiment.In a preferred embodiment, fluorescence intensity can be removed background signal by GenePix Pro 3.0 softwares, analyzes then.Based on size and " sign " (data of losing) of path intensity, point, expression data is filtered then, and all genes of each array are calculated Cy5/Cy3 ratio and carry out normalization method with the intermediate value for the center correlative value.To the arbitrary specific hybridization site of transcript array, can calculate the emission ratio of two kinds of fluorophores.Ratio does not rely on homogenic absolute expression levels, but to those expression be subjected to administration, genetically deficient or other any incidents obviously regulation and control gene of great use.

The relative abundance of certain mRNA in the method according to this invention, two biological samples can be used as the scoring (being that the concentration of mRNA in two kinds of test sources is different) of disturbance and level of disruption, or thinks do not have disturbance (that is, relative concentration equates).In different embodiment, two RNA sources differ at least one factor at least about 25% (the RNA abundance many 25% in the another kind of source of the relative abundance of a kind of RNA of source), more generally have 50% approximately, this factor even differ about 2 times (twice abundance) more generally, when 3 times (3 times of abundance) or 5 times (5 times of abundance), this difference is cited as disturbance.

Preferably, except identifying that disturbance is the positive or the feminine gender, it is favourable measuring the turbulent size.This can carry out as mentioned above, as by calculating the emission ratio between two kinds of fluorophores that are used for the difference mark, or by the conspicuous similar method of those skilled in the art.

8. pathway response and gene expression profile

In one embodiment of the invention, measure gene expression profile by the gene expression profile of observing clinical sample of interest.In one embodiment of the invention, by with two kinds of not probe mixture and microarray hybridizations of isolabeling, can set up the dna microarray of reflection biological sample transcriptional state interested, wherein the mRNA of corresponding a kind of clinical interested sample of each probe or standard model.According to the present invention, two kinds of samples are same kinds, promptly same strain and types of organization, but can be different in clinical diagnosis.Those genes of expressing height correlation can belong to a kind of gene expression profile.

In addition, for reducing experimental error, be preferably in the double-colored differential hybridization experiment and exchange two kinds of fluorescent markers to reduce deviation to each gene or array site.In other words, preferably (promptly earlier with the mRNA genetic expression of two kinds of tested cells of a kind of marking method measurement, with a kind of fluorophore mark by the turbulent cell, with second kind of fluorophore mark not by the turbulent cell), measure two kinds of gene expression of cells (promptly with opposite labelling method then, with second kind of fluorophore mark by the turbulent cell, with first kind of fluorophore mark not by the turbulent cell).The a plurality of observed values that surpass exposure levels and disturbance control parameters level can provide extra experimental error contrast.If fully sampling when the width of selecting spline function S when (being used at reaction functions interpolation response data between mean error and structure are lost), just can realize exchanging.

9. other method of measuring of transcriptional state

The transcriptional state of cell can be measured by other gene expression technique.The restriction fragment that in these technology some produce many finite complexity is used for electrophoretic analysis, the method that combines as the digestion of two restriction enzymes and stage primer (referring to, as European patent 0534858 A1, on September 24th, 1992 is by people such as Zabeau application), or the method for the restriction fragment of the most approaching described mRNA end in selection site (referring to, as people such as Prashar, 1996, Proc.Natl.Acad.Sci.USA93:659-663).Other method can be carried out the statistics sampling to the cDNA pond, as by each cDNA is measured abundant base (as, 20-50 base) determine each cDNA, or the sequence (as 9-10 base) of survey short label, wherein this label be produce with respect to the known site of a certain mRNA end (referring to, as Velculescu, 1995, Science 270:484-487).

10. the otherwise mensuration of biological aspect

In a plurality of embodiment of the present invention, can measure biological state except transcriptional state, as translation state, active condition or combination, so that acquisition is to the reaction of medicine and path.The details of these embodiment has description hereinafter.

11. the embodiment that transcriptional state is measured

The measurement of transcriptional state can be carried out according to several different methods.Such as, to proteic full genome monitoring (that is, " protein groups ", people such as Goffeau, the same) can realize that binding site wherein comprises, and numerous albumen immobilized, that the pair cell genome is coded have specific antibody (preferred monoclonal antibody) by making up microarray.Preferably, the antibody of existence can be at the major part of proteins encoded, or at least at those albumen relevant with medicine interested.The method for preparing monoclonal antibody be know (referring to, as Harlow and Lane, 1988, Antibodies:A Laboratory Manual, Cold Spring Harbor, the N.Y. document is incorporated herein by reference in full).In a preferred embodiment,, and produce the monoclonal antibody that resists these peptide sections according to the genome sequence design section of synthesized peptide of cell.With such antibody array, cell protein is contacted with array, with regard to available analytical procedure known in the art analyze they in conjunction with situation.

Perhaps, albumen can separate by the two-dimensional gel electrophoresis system.Two-dimensional gel electrophoresis is known in this area.Typical two-dimensional gel electrophoresis comprises along the unidimensional isoelectrofocusing, then along the second SDS-PAGE electrophoresis of tieing up.Referring to, as people such as Hames, 1990, Gel Electrophoresis of Proteins:A Practical Approach, IRL Press, NewYork; People such as Shevchenko, 1996, Proc.Nat ' l Acad.Sci.USA93:1440-1445; People such as Sagliocco, 1996, Yeast 12:1519-1533; Lander, 1996, Science 274:536-539.The electrophoretogram that forms can be by the multiple technologies analysis, comprises mass-spectrometric technique, with monoclonal antibody and many anti-carry out western blotting and immunoblotting assay, and inner and N-end micrometering preface.Use these technology, can identify all proteic big fragments that under given physical condition, produce, be included in cell that medicine contacts in (as in yeast) or by disappearance or cross and express all proteic big fragments that produce in the cell that specific gene modifies.

12. embodiment based on the biological aspect others

Although method of the present invention is to set forth by the example of gene expression pattern, method of the present invention can be used for any cellular constituent that can be monitored.

Particularly, with certain disturbance proteins associated activity (as drug effect) can be determined the time, embodiments of the invention just can be based upon on these based measurement.Determination of activity can be by any active function of surveying that is applicable to, method biochemical or physics is carried out.When activity comprised chemical transformation, cell protein can contact with natural substrate and measure velocity of variation then.When activity comprise between the poly unit in conjunction with the time,, can measure protein-bonded quantity or measure the second level outcome that causes in conjunction with the back in conjunction with combining between mixture and the DNA as activatory DNA, as the mRNA quantity of transcribing.If having only a kind of functionally active is known (as in cell cycle regulating), behavior performance that so can overview function.No matter be known or mensuration, the variation of protein-active can constitute response data, and these data can be analyzed by method of the present invention.

In another non-restrictive example, response data can be that the confounding factor of cell biological state constitutes.Response data can comprise as the variation of the variation of certain mRNA abundance, certain albumen abundance and the variation of certain protein-active.

II. proteome analysis

On the other hand, the invention provides the method for certification mark thing, these marker differences are present in transitivity HCC tumor sample or have in HCC susceptibility patient's (that is, very easily develop into HCC but also do not find the patient of tumour) the tissue sample.These markers can detect in multiple biological sample.Sample is the lysate of biological tissue samples preferably.

Any appropriate means all can be used to detect one or more described markers herein.For example, available gas phase ion spectrometry assay method.This technology comprises, as laser desorption/MALDI-MS assay method.Preferably, sample prepared before the gas phase ion spectrometry assay method, as by presorting level a separation, two-dimentional gel chromatography, high performance liquid chromatography etc., so that help the certification mark thing.Method certification mark thing outside the available gas phase ion spectrometry assay method.For example, the marker in the application immunoassay monitoring sample.These detection methods have a detailed description hereinafter.

A. gas phase ion spectrometry assay method

Marker in the biological sample can detect (mass spectroscopy is better) with the gas phase ion spectrometry assay method.In one embodiment, can use substance assistant laser desorpted/ionization (" MALDI ") mass spectrometry.In another embodiment, can application surface strengthen laser desorption/ionization (" SELDI ") mass spectrometry.

1. the specimen preparation before gas phase ion spectrometry is measured

The available a kind of or multiple standard technique well known in the art of coupling prepares sample, with monitoring and the evaluation of further assistance to marker in the sample.Such as, before gas phase ion spectrometry determination and analysis method, can sample classification be separated obtaining less composite sample with the method below one or more: size exclusion chromatography, anion-exchange chromatography, affinity chromatography, order extraction, gel electrophoresis, high performance liquid chromatography (HPLC).

Marker also can improve its resolving power or determine its identity through modifying before analysis.Such as, can be before analysis with the marker proteolytic digestion.Digest the fingerprint that the fragment that obtains can be used as marker with suitable proteolytic enzyme (as pancreatin), can realize indirect detection them.

2. sample contacts laggard promoting the circulation of qi ion spectra assay method analysis mutually with a kind of substrate

Biological sample can contact with substrate, as is applicable to the spectrometry probe of gas phase ion spectrometry determinator.Perhaps, substrate can be a kind of independently material, and it can be placed on the spectrometry probe that is applicable to the gas phase ion spectrometry determinator.

Spectrometry instrument probe can be any suitable shape, if it can on the gas phase ion spectrometry determinator, use (as, insert the gas phase ion spectrometry determinator removedly).Spectrometry instrument probe substrate can be made with any suitable solid or porous materials.The spectrometry instrument probe that is applicable to embodiments of the invention has description as U.S. Patent No. 5,617 among 060 (Hutchens and Yip) and the WO 98/59360 (Hutchens and Yip).

If the complicacy of sample has fully reduced as said quilt above, sample can contact with the substrate that any gas phase ion spectrometry determinator is suitable for.Before carrying out the gas phase ion spectrometry determination and analysis, on the marker of substrate surface, generally can use a kind of energy absorption molecule (" EAM ") or substrate material.The energy absorption molecule can contact under any suitable state with the sample that contains marker.

The complicacy of sample can further reduce with substrate, this substrate contain can with one or more marker bonded sorbent materials.The sorbent material of binding label (as continuous or discontinuous mode) in any suitable manner is applied to substrate, sample also can contact under any suitable state with the substrate that contains sorbent material, as water-bath, soak into, flood, spray, splash or move liquid etc.After the contact, the best unconjugated material of flush away substrate surface, thus make substrate surface only stay the bonded material.

3. desorb/ionization and detection

The marker of substrate surface can be measured at gas phase ion spectrometry and go absorption and ionization in the art.Any suitable gas phase ion spectrometry determinator can both use, as long as it can make the marker on the substrate dissociate.The gas phase ion spectrometry determinator preferably can be to the marker quantitative analysis.In one embodiment, the gas phase ion spectrometry determinator is a mass spectrograph, preferably the laser desorption time-of-flight mass spectrometer.In another embodiment, available ion diffusion spectrometry instrument certification mark thing.In another embodiment, total stream of electrons measuring apparatus can be used for detecting and the identifying mark thing.

4. data analysis

The data that obtain by desorb and certification mark thing can be with any appropriate means analysis.In one embodiment, with a programmable digital computer analytical data group.Computer program generally comprises a readable media, is used for storage code.Some code is exclusively used in memory, comprising each characteristic site on the spectrometry instrument probe, at the kind of the sorbent material in this feature site and the elution requirement of wash-out adsorptive.Computer comprises these codes simultaneously, and they are as the data of input.The strength of signal of each molecular grouping is from addressable position specific on the probe.The quantity of the detected marker of these data representations comprises the intensity of the signal that each marker generates.

Data analysis can may further comprise the steps, and measures the strength of signal (as peak value) of tested marker and removes the data that depart from default statistical distribution.The peak value normalization method of observing, this is a process of calculating each peak heights with respect to a certain reference.Such as, with reference to being the background noise that instrument and chemical substance (as endergonic molecule) are produced, generally be made as zero.Then, each marker that detects or the strength of signal of other biomolecules go up at required scale (as 100) to be represented with relative intensity.Perhaps, establish a standard (as serum protein) at sample, base peak can be used as the relative signal intensity of each detected marker of reference calculation or other markers.

Computer can be different display formats with the data conversion that obtains.In the form of a kind of being called " spectrum picture or keep collection of illustrative plates ", collection of illustrative plates that can display standard, wherein image table is understood the quantity at the marker of each specific molecular weight that arrives probe.Be called in the form of " peak collection of illustrative plates " at another, spectrum picture only keeps peak height and quality for information about, and the image of formation is fairly simple to be understood, can the approaching marker of easier resolution molecular weight.Be called in the form of " gel images " at another, each quality of peak collection of illustrative plates is converted into gray level image on the basis of each peak height, looks similar with the band on the running gel.Be called in the form of " three-dimensional overlapping " at another, can several spectrograms are overlapping to compare their minute differences in relative peak heights.Be called in the form of " difference spectrum " at another, can more two or more wave spectrums, more given prominence to the marker that is upward or downward between different markers and the sample.The marker pattern (wave spectrum) of any two samples can compare on a macro scale.In another form, can adopt igniting scatter diagram (Spotfire Scatter Plot), wherein tested marker marks with point in the drawings, and wherein figure axle is represented the apparent molecule of tested marker, the strength of signal of the tested marker of another representative.The quantity of marker all is kept in the computer-readable medium in the tested marker of each biological sample and the sample.These data can with compare (as detected marker collection of illustrative plates or quantity in contrast, for example the patient of also nd transitivity HCC of sample or HCC susceptibility).

Predict HCC patient's metastatic potential or have chronic hepatitis patients to develop into the method for the possibility of HCC, can come specific implementation by digital machine run time version, and this data set is to derive from and chip signal after patient's sample contacts with process data set.The purpose that code is carried out by digital machine is in order to create analytical model.This code can be write with any suitable computer programming language, and these programming languages have Visual Basic, Fortran, C, C ⁺⁺Deng.Digital machine can be to use arbitrary standard or specialized operating system, as operating system, miniature, the mini or large-scale computer based on Windows.Standard PC (PC) can be come the execution analysis method according to embodiments of the invention.

B. measure by immunoassay

Immunoassay can be used to detect with analyzing samples in marker.This method is made up of following: (a) provide the energy specific combination in the antibody of marker; (b) antibody is contacted with sample; (c) existence that detects the antibody complex that is incorporated into marker in the sample whether.

Preparation can with the polyclone and the monoclonal antibody method of cell marker generation specific reaction, be well known by persons skilled in the art.Consult document Coligan, Current Protocols in Immunology (1991); Harlow ﹠amp; Lane, Antibodies:A Laboratory Manual (1988); Goding, Monoclonal Antibodies:Principles and Practice (2d ed.1986); And Kohler ﹠amp; Milstein, Nature 256:495-497 (1975).For example, how anti-in order to prepare, the target protein of purifying is mixed mutually with adjuvant, be used for immune animal then.After the target protein antibody of high titre produces, collect blood from animal, the preparation antiserum(antisera) is used for immunoassay.For the preparation monoclonal antibody, will make unlimited line of breeding with the animal splenocyte of target protein immunity, this is usually by merging (consulting Kohler and Milstein, Eur.J.Immunol., 6:511-519,1976) with the myeloma cell.According to whether producing the antibody that target protein is had expection specificity and affinity, to screening by the cell clone that single permanent cell produced.

If marker is not a known protein in the database, even have only the part of marker, available this knowledge is determined nucleic acid and aminoacid sequence.For example, based on the sequence of marker N terminal amino acid, prepare the probe of degeneracy.Come screening-gene group or cDNA library with these probes then, wherein this library is to create with the sample that originally detects marker.Use known technology, positive colony can be identified, amplification, and its recombinant DNA sequence can be by subclone.Consult, for example, people such as Ausubel, Current Protocols for Molecular Biology, 1994 andSambrook and Russell, the same.Based on the oligonucleotide of coded markings thing, the antibody of anti-marker can prepare with any appropriate method known in the art.For example consult people such as Huse, Science 246:1275-1281 (1989); People such as Ward, Nature 341:544-546 (1989).

After antibody is provided, marker can detect with suitable immune combination technology and/or quantitatively (referring to, for example, U.S. Patent No. 4,366,241; 4,376,110; 4,517,288; With 4,837,168).The available analysis comprises: as enzyme immunoassay (EIA) as enzyme linked immunosorbent assay (ELISA), reflectivity immunoassay (RIA), Western engram analysis, slit spot analysis.These methods are at Methods in Cell Biology:Antibodies in Cell Biology, vol 37 (Asai edits .1993); Basic and Clinical Immunology (Stites ﹠amp; Terr, eds., 7th ed.1991); With Harlow ﹠amp; Among the Lane (the same) narration is arranged.

The diagnosis of C, transfevent HCC or HCC susceptibility

On the other hand, the invention provides a kind of method, the one or more markers of this method by having identified among the use table 2-7 develop into transfevent tumour possibility or chronic hepatitis patients to HCC patient and change HCC trend into and make diagnosis.Although lack to the marker that has only from table 2-7 marker, to select, also can make correct diagnosis, a plurality of markers of preferred use are because multi-tracer can obtain how reliable result.Preferably, at least 10 cell markers are comprised in marker and concentrate in the table 2, and be used to predict HCC patient's metastatic potential, for example more preferably in the table 2 at least 15,20,25,30,40,50,60,70,80 90 or 100, even most preferred all 153 markers are used as marker.Similarly, more preferably have 15,20,25,30,40,50,60,70,80 90 or 100 in the table 5 at least, even most preferred all 273 markers are used as marker, are used to measure the risk that chronic hepatitis patients is suffered from HCC.The marker of showing to have identified among the 2-7 can use separately, the marker coupling in also can showing together in other forms, or with diverse marker coupling, so that assist diagnosis trouble transfevent HCC or chronic hepatitis patients to develop into the susceptibility of HCC.Compare with the patient tissue sample of non-transfevent HCC and no HCC susceptibility, in transfevent HCC sample or HCC susceptibility patient tissue sample, the marker among the table 2-7 is that difference exists respectively.For example, compare with the patient tissue sample of non-transfevent HCC and no HCC susceptibility, some marker high level expressions in and/or upper frequency come across in transfevent HCC or the HCC susceptibility patient tissue sample.Therefore, one or more such markers in the human body, the useful information that can provide someone to suffer from transfevent HCC or easily suffer from the possibility aspect of HCC.

Therefore, the example of invention comprises the method for assistant analysis diagnosis HCC metastatic potential, and assistant analysis diagnosing chronic liver problem sufferer develops into the method for HCC possibility, wherein this method comprises: (a) detect at least one mark in sample, this mark is selected from the mark that table 2-7 has identified; (b) mark with one or more detections is associated with the possibility that diagnosis or the liver problem sufferer of transitivity HCC develop into HCC.This dependency can be considered and the marker quantity of the contrast amount of mark (as non-transfevent HCC or do not have the individuality of HCC susceptibility) when comparing in the sample.Dependency can consider whether the appearance of mark in the sample to be checked detects frequency with same mark in control sample.Dependency can be taken into account these factors, whether suffers from transitivity HCC and suffers from the serious hepatopathy that may develop into HCC so that judge someone.

The appropriate samples that is used for detecting mark can be from arbitrary individual acquisition.Preferably, sample is the hepatic tissue sample that obtains from individuality.If desired, sample can prepare according to the method described above to strengthen the detectability of mark.

Can adopt any proper method to come mark in the test sample.Such as, can adopt the gas phase ion spectrometry assay method as mentioned above.Use these methods, can detect one or more marks.Preferably, whether test sample exists a plurality of marks.Detect the existence of a plurality of marks rather than single mark, can provide more information for diagnosis.In particular, a plurality of marks of detection can increase true positives and the true negative in the diagnosis in a sample, will reduce false positive and false negative in the diagnosis simultaneously.

Then, the detected result of mark is associated with the possibility that develops into transfevent HCC, perhaps develops into the HCC susceptibility with serious liver problem sufferer and is associated.In some example, only detect whether mark exists and non-quantitative mark quantity is exactly useful, and can with develop into the general diagnostic result that transfevent HCC or serious liver problem sufferer develop into the susceptibility of HCC and be associated.

In addition, detect mark and can comprise quantitative marker, and with the marker detection result with develop into transfevent HCC or seriously liver problem sufferer's general diagnostic result of developing into the susceptibility of HCC be associated.For example, detected OPN level increase among the transfevent HCC patient.Like this, if the mark amount of certain individuality to be checked is higher than normal amount, this individuality has the height possibility to develop into transfevent HCC or the tendency that develops into HCC is arranged for serious liver problem sufferer so.

When the mark quilt is quantitative, can be compared with the control.Contrast can be, the mark mean value in the similar sample of normal individual for example, and wherein this normal individual does not have the tendency that develops into transfevent HCC, or does not have the tendency that develops into HCC for chronic hepatitis patients.Control group quantity is measured under identical or similar substantially experiment condition with sample size to be checked.For example, if sample to be checked is certain individual serum sample and is to adopt specific probe to detect certain mark that the same probe of contrast quantity advantageous applications of this mark is measured patient's serum sample so.Preferably, the contrast quantity of mark is at the sample of the normal individual that a large amount of no HCC shift or do not have on the basis of tissue sample of individuality of HCC susceptibility and determine, so that be reflected in the variation of mark quantity among this crowd.

Computer software can be analyzed the data that mass spectrum records.The code of this software can be computer-readable form with the mass spectroscopy signal transition.Whether this software also can comprise code, and this code is used to use algorithm and analyzes aforementioned signal, to have represented in clear and definite this signal corresponding to mark of the present invention or other useful mark signals " peak ".This software also can comprise code, and this code is used for execution algorithm, thereby the type signal feature of specimen signal with the serious hepatopathy patient of " normally " and transfevent HCC or HCC susceptibility compared, and determines the affinity between two signals.This software also can comprise code, and this code points out sample to be checked the most approaching any situation, and general diagnosis is provided.

III. treat the adjusting of target spot biologic activity

Osteopontin (OPN) and EpCAM and HCC patient's transfer and chronic hepatitis patients develop into HCC and all are proportionate.Therefore, one object of the present invention is differentiated adjusting exactly, especially suppresses the active compound of OPN or EpCAM.

A. the mensuration of biological function

OPN and allelotrope thereof and multiple variation thing all are secretion property phosphorproteins, and it is shown in SEQ ID NO:2 by SEQ ID NO:1 coding and its aminoacid sequence.Can adopt in the multiple body and external method is measured function, chemistry and the physical action of OPN polypeptide, as measuring receptors bind (as combine) etc. with radioreceptor, thus the activity of evaluation OPN polypeptide.More the incident in downstream (as changing such as cell incidents such as cell fission, cytodifferentiation) also can be used as the active change of secondary indication OPN.In addition, these methods can be used to detect and the active antagonist of screening OPN.Antagonist can change the form of OPN from the gene angle, as the negative form of proteinic dominance.The active antagonist of these OPN can be used for treating transfevent HCC.

The optional own SEQ ID NO:2 polypeptide of sequence of the OPN that is used to analyze, or its conservative varient or fragment of modifying.At large, the homogeny at least 70% of aminoacid sequence, randomly at least 80%, or 90-95% at least randomly.Randomly, the polypeptide that is used to analyze can comprise the OPN structural domain, as receptor binding domains, extracellular matrix in conjunction with territory etc.OPN or its structural domain can be covalently bonded in heterologous protein to be formed for the chimeric protein of this analysis.

Adopt aforesaid reorganization or natural OPN polypeptide, can test the active conditioning agent of OPN.This protein can be recombinated or natural form is expressed in cell, secretion from cell, express in tissue or animal, and is and separated.For example, can use liver section, isolating liver cell or cell transformed.Adopt in a kind of body as herein described or in vitro method can detect antagonism to OPN.In addition, the proteic receptor binding domains of available OPN detects receptors bind in external liquid phase or solid state reaction.

Acceptor combines with OPN, structural domain or chimeric protein, can be in solution, on the bilayer capsule, on the solid phase carrier, test on the lipid monolayer or on the vesicle.But the variation of application of spectral characteristic (as fluorescence, absorbancy, specific refractory power), fluid (as form), chromatography or dissolution characteristics test antagonist in conjunction with situation.

With sample or the analysis that potential OPN inhibitor is handled,, check the antagonism degree by comparing with the control sample that does not contain compound to be checked.It is 100 that control sample (not handling with antagonist) is decided to be relative OPN activity value.When compared with the control, the OPN activity value be about 90%, randomly 50%, randomly during 25-0%, just think and realized antagonism OPN.

Under the situation that antagonist exists, can pass through the variation of the binding ability of detection OPN and Vitronectic receptor, assess the change of OPN receptors bind.In a word, the scope of compound to be checked is that 1pM is to 100mM.

Compound to be checked can be measured by measuring above-mentioned arbitrary parameter the influence of polypeptide function.Any active corresponding physiology of OPN that influences changes, and can be used to estimate the influence of compound to be checked to polypeptide of the present invention.When using intact cell or zoometry function as a result the time, people also can measure various effect, as known and unknown genetic marker transcribe variations (as the Northern trace), as the variation (growing or the pH variation) of cellular metabolism as cell.

Similarly, can on aforesaid same principle and methodological basis, monitor the biological function of EpCAM.For example, known EpCAM plays a role in the adhesion of epithelium source sexual cell, and its normal function relies on the outer and born of the same parents' intracellular domain of its born of the same parents.Therefore, can based on such as cell aggregation, with its known interruption that combines the specific action (as by born of the same parents' intracellular domain and Actin muscle effect) and the signal transduction of counterpart (known this is that EpCAM regulates), detect the function of EpCAM.Various cell incident can be used as the active indicator of EpCAM, and helps to screen the compound as the EpCAM antagonist.

A. antagonist

As OPN or EpCAM antagonist and the compound of testing can be any little chemical substance, or biological substance, for example albumen, sugar, nucleic acid or lipid.Anti-proteic different antibodies is possible antagonist material standed for.For example, many monoclonal antibodies, as 17-1A and GA733, having known can be specifically in conjunction with EpCAM, thereby can test the ability that their disturb EpCAM biological function by appropriate analysis.

In addition, antagonist can be that the OPN of change or the form of EpCAM are gone up in heredity, for example so-called " dominance feminine gender (dominant negative) " form, the active form of a kind of lifeless matter, it is by competing the normal function that limited binding partners suppresses the wild-type copy.Usually, test compounds is chemical small molecules and peptide.Though compound is dissolved in the aqueous solution or organic solvent (particularly based on DMSO's) more, any basically compound can be used as the potential antagonist in the analytical procedure of the present invention.The compound of conveniently originating from any is provided by the testing process automatization with for analysis, this analysis method is designed to screen big chemical libraries, usually analyze is to carry out parallel (for example in detecting automatically, adopting the titrating form of micropore on microwell plate) of carrying out.Should be understood that many compound suppliers, comprise Sigma (St.Louis, MO), and Aldrich (St.Louis, MO), Sigma-Aldrich (St.Louis, MO), Fluka Chemika-BiochemicaAnalytika (Buchs Switzerland) or the like.

In a preferred examples, the method for high flux screening comprises chemical libraries or the peptide storehouse that a combination is provided, and this storehouse comprises a large amount of potential curative compounds (potential conditioning agent or ligand compound) that have.As used herein, be somebody's turn to do " combinatorial chemical library " or " part storehouse " by one or more analytical procedure screenings, have the active library member of required feature (concrete chemical species or subgroup) thereby identify.The compound that identifies like this can be used as conventional " lead compound ", perhaps itself can be used as potential or actual therapeutical agent.

Combinatorial chemical library is the integrated of different compounds, can be by chemosynthesis or biosynthesizing, and " building block (the building blocks) " of comprehensive many chemistry is as reagent.For example, the chemical libraries of linear combination (as peptide library) is for given compound length (as the amino acid quantity of polypeptide compound), forms by combination one group of chemistry building block (amino acid) on arbitrary possibility direction.This built-up type by chemical building block mixes, and just can synthesize up to a million kinds of chemical compounds.

How preparing and screen combinatorial chemical library, is well known to those skilled in the art.These combinatorial chemical libraries comprise that (but being not limited to) peptide library (sees United States Patent (USP) 5,010,175; Furka, Int.J.Pept.Prot.Res.37:487-493,1991; With people such as Houghton, Nature 354:84-88,1991).The chemical process in other manufacturing chemistry diversity storehouse also can be used.These chemical processes comprise (but being not limited to): class peptide (as: PCT publication No.WO 91/19735), encoded polypeptides (as: PCT publication WO 93/20242), biological oligomer at random (as: PCT publication No.WO 92/00091), the benzodiazepine class is (as U.S. Patent number 5,288,514), various body (diversomer) is hydantoins for example, benzodiazepine class and dipeptide (people such as Hobbs, Proc.Nat.Acad.Sci.USA 90:6909-6913,1993), divinyl polypeptide (people such as Hagihara, J.Amer.Chem.Soc.114:6568,1992), polypeptide stand-in (people such as Hirschmann with non-peptide class of glucose skeleton, J.Amer.Chem.Soc.114:9217-9218,1992), the simulation organic synthesis storehouse of little compound (people such as Chen, J.Amer.Chem.Soc.116:2661,1994), oligomerization carboxylamine (people such as Cho, Science 261:1303,1993), and/or peptide acyl phosphonic acid ester (people such as Campbell, J.Org.Chem.59:658,1994), nucleic acid library is (referring to Ausubel, Berger and Sambrook, all the same), the peptide nucleic acid(PNA) storehouse (referring to, for example, United States Patent (USP) 5,539,083), antibody library (referring to, for example, people such as Vaughn, Nature Biotechnology, 14 (3): 309-314,1996 and PCT/US96/10287), the carbohydrate storehouse (referring to, for example, people such as Liang, Science 274:1520-1522,1996 and United States Patent (USP) 5,593,853), little organic molecule library (referring to, for example, benzodiazepine, Baum C﹠amp; EN, January 18, p33,1993; Isoprenoid, United States Patent (USP) 5,569,588; Thiazole and three polythiazoles, United States Patent (USP) 5,549,974; Tetramethyleneimine, United States Patent (USP) 5,525,735 and 5,519,134; Morpholinium compound, United States Patent (USP) 5,506,337; Benzodiazepine, 5,288,514, or the like).

The preparation combinatorial libraries equipment commercialization (referring to, for example, 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050Plus, Millipore, Bedford, MA).In addition, many combinatorial libraries also commercialization (referring to, for example, ComGenex, Princeton, N.J., Tripos, Inc., St.Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

The high throughput analysis of C. solid-state and solubility

In one embodiment, the invention provides the soluble analysis method, wherein use molecule, for example structural domain (as receptors bind structural domain, extracellular matrix binding domains or the like); Covalency is connected in heterologous protein to form the structural domain of chimeric molecule; OPN or EpCAM; The cell or tissue of natural or recombinant expressed OPN or EpCAM.In another embodiment, the invention provides the analyzed in vitro method based on the high-throughput form of solid phase, wherein the cell or tissue of structural domain, chimeric molecule, OPN or EpCAM or expression OPN or EpCAM is attached to solid phase carrier matrix.

In high throughput analysis of the present invention, can screen up to thousands of different antagonists in one day.Particularly, the hole of each microwell plate can be at selected potential conditioning agent separate analysis, and perhaps, if consider the influence of concentration or incubation time, each conditioning agent can detect with 5-10 hole.Therefore, the microwell plate of a standard can detect about 100 (as 96) and plant conditioning agent.If use the plate in 1536 holes, a plate is easy to analyze 100 to 1500 different compounds so.If use whole system of the present invention, may analyze several different plates every day, may screen and reach 6,000-20,000 different compound.Recently, (Palo Alto, CA) company has developed little liquid mode of reagent operation to Caliper Technologies.

Molecule (s) of interest can be directly or indirectly be connected on the solid phase composition by mode of connection (as passing through label) covalently or non-covalently.Label can be various composition.Usually, the molecule (label combination) that combines label is fixed on the solid phase carrier, and the interested molecule (as interested signal transducers) that combines label is connected in solid phase carrier by the interaction with label and label binding substances.

Based on the interaction of molecules of fully describing in the document, can use many kinds of labels and binding substances thereof.For example, when label has such as natural binding substancess such as vitamin H, albumin A or Protein G, it can with suitable label binding substances (affinity element, streptavidin, neutral affinity element, immunoglobulin Fc section or the like) coupling.Be coupled to the antibody of molecule with natural binding substances (as vitamin H), be well-off and be suitable label binding substances; See SIGMA Immunochemicals1998 catalogue (SIGMA, St.Louis MO).

Similarly, any haptens or antigenicity compound can be used for forming label/label binding substances pairing with suitable antibodies.The commercialization of thousands of specific antibodies, and a lot of extra antibody has description in the literature.For example, in common type, label is that first antibody and label binding substances are the two anti-of identification first antibody.Except antibody-antigenic interaction, the receptor-ligand interphase interaction also is suitable as label and the label binding substances is right.For example, (cell receptor-ligand interaction for example is as Transferrins,iron complexes, c-kit, virus receptor part, cytokine receptor, chemokine receptors, interleukin-2-receptor, immunoglobulin receptor and antibody, calcium conglutnin family, integrin family, select plain family or the like for cell-membrane receptor wedding agent and antagonist; For example see Pigott ﹠amp; Power, The Adhesion Molecule Facts Book I (1993)).Similarly, toxin and venom, the epitope of virus, hormone (as opium, steroid or the like), intracellular receptor (as mediating the acceptor of different little part effects, these little parts comprise steroid, Triiodothyronine, retinoid and vitamins D, polypeptide), medicine, phytohemagglutinin, sugar, nucleic acid (linearity or ring-type polymer structure), oligosaccharides, albumen, phosphatide and antibody can both interact with various cell receptor.

The synthetic polymer also can form suitable label or label binding substances as polyurethane, polyester, poly-carbon ester, poly-urea, polymeric amide, polymine, poly-sulfuration vinylbenzene, polysiloxane, polyimide and poly-acetic ester.Many labels/label binding substances pairing also can be used for analytical system as herein described, and this is conspicuous for having read the technician after the disclosure.

Linker such as peptide, polyethers and analogue commonly used also can be used as label, and comprise peptide sequence, according to appointment the amino acid whose polyglycine sequence of 5-200.To those skilled in the art, these flexible linkers all are known.For example, poly-(ethylene glycol) linker can be from Shearwater Polymers, Inc.Huntsville, and Alabama buys.These connectors can randomly contain amido linkage, sulfydryl key or exclusive-OR function key.

The label binding substances can be with existing various different fixations on solid-phase matrix.Usually, make solid-phase matrix derivatize or functionalization by all or part of matrix is exposed to chemical reagent, wherein this chemical reagent is fixed in the surface with chemical group, and this chemical group can react with the part of label binding substances.For example, be fit to connect long-chain group partly and comprise amine, hydroxyl, sulfydryl and carbonyl.Aminoalkyl group silane and hydroxyalkyl silane can be used for activating kinds of surface, as glass surface.The structure of this kind solid phase biological polymer array has abundant description in the literature.For example see Merrifield, J.Am.Chem.Soc.85:2149-2154 (1963) (having described solid phase synthesis) such as materials such as peptides; People such as Geysen, J.Immun.Meth.102:259-274 (1987) (having described synthetic solid phase composition on needle point); Frank ﹠amp; Doring, Tetrahedron44:60316040 (1988) (having described synthetic different peptide sequence on cell); People such as Fodor, Science, 251:767-777 (1991); People such as Sheldon, Clinical Chemistry 39 (4): 718-719 (1993); With people such as Kozal, Nature Medicine2 (7): 753759 (1996) (all having described the biological polymer array that is fixed in solid-phase matrix).The method non-chemically that the label binding substances is fixed in matrix comprises: heating, crosslinked or the like by uviolizing.

D. computer based analysis

The method that OPN or the active compound of EpCAM are regulated in another kind of screening is computer assisted medicinal design, and wherein the structural information that produces based on aminoacid sequence utilizes computer to produce the three-dimensional structure of OPN or EpCAM.The aminoacid sequence of input directly and the algorithm of having set up by computer program energetically produces secondary, three grades and quaeternary protein structure model.Then, check these protein structures, to confirm having in conjunction with active structures zone (as being incorporated into part).These zones are used to differentiate and are incorporated into proteinic part.

By at least 10 amino-acid residues of input or the OPN of corresponding encoded or the nucleotide sequence of EpCAM polypeptide in computer system, can produce proteinic 3 d structure model.For example, the nucleic acid of OPN amino acid sequence of polypeptide or this polypeptide of encoding is selected from SEQ ID NO:1 or 2, and the conservative property modified forms.Aminoacid sequence has been represented proteinic primary sequence or subsequence, its proteinic structural information of having encoded.By at least 10 aminoacid sequence residues of computer keyboard input (or nucleotide sequence of coding ten amino acid), computer-readable medium includes but is not limited to: electronic storage medium (as disk, tape, magnetic holder and chip), optical medium (as CD ROM), internet website information releasing or pass through RAM.Then, use software well known by persons skilled in the art,, can produce proteinic 3 d structure model by the unify interaction of aminoacid sequence of department of computer science.

Aminoacid sequence is a primary structure, and it has been encoded and has formed secondary, three grades and the required information of quaternary structure of proteins of interest.Software is watched some parameter by the primary sequence coding that produces structural models.These parameters are called as " energy term ", mainly comprise electrostatic potential, hydrophobic potential, the accessible surface of solvent and hydrogen bond.The secondary energy term comprises Van der Waals force.The structure that biomolecules forms reduces energy term with accumulating form.Therefore, computer program can utilize primary structure or amino acid sequences encoded energy term to produce the secondary structure model.

Then, based on the energy term of secondary structure, form by the coded tertiary structure of secondary protein structure.At this moment the user can the amount of imports outer parameter, for example albumen whether be film in conjunction with or soluble, location in vivo, cellular localization (as tenuigenin, surface, nuclear).These variablees can form the tertiary structure model in conjunction with the energy term of secondary structure.When the simulation tertiary structure, computer program mates the hydrophobicity face of secondary structure mutually, and the wetting ability face is mated mutually.

In case the generation compound structure, the protein ligands calmodulin binding domain CaM can be discerned by computer system.The three-dimensional structure of potential part can generate by the input amino acid of compound or nucleotide sequence or chemical formula, as mentioned above.The three-dimensional structure of this potential part is compared with OPN or EpCAM albumen, can determine bonded part with OPN or EpCAM.The affine of albumen and part determines that in conjunction with the available energy quantifier any part and protein bound possibility are bigger.

Computer system also is used to screen homologue between mutant, polymorphic variation's body, allelotrope and the kind of OPN gene or EpCAM gene.These mutant are relevant with symptom or hereditary property.As mentioned above, gene chip can be used for screening homologue between mutant, polymorphic variation's body, allelotrope and kind with similar technology.In case varient is identified, diagnostic method can be used to differentiate patient with this mutator gene.For example, differentiate that the OPN gene of sudden change comprises first aminoacid sequence that is selected from SEQ ID NO:1 and 2 of accepting input or the nucleotide sequence of encoding OPN, and the conservative property modified forms.As mentioned above, sequence is imported computer system.Then, first nucleic acid or aminoacid sequence are compared with second nucleic acid or aminoacid sequence, wherein second sequence and first sequence are basic identical.As mentioned above, with second sequence input computer system.In case after first and second sequences were contrasted, Nucleotide or amino acid different between sequence just were identified.These sequences can be represented the equipotential difference of OPN gene, and the sudden change relevant with illness and hereditary property.Same general policies also can be used for detecting EpCAM varient and mutant.

D. test kit

Proteins of interest and homologue thereof are the effective tools of determining antagonist.For example, with the OPN-specificity substance (as OPN probe and primer) of OPN nucleic acid specificity hybridization, and with the OPN specificity substance (as OPN antibody) of OPN albumen specific combination, can be used for detecting liver cell expression, the signal conduction is regulated and HCC shifts diagnosis.Identical universal method is suitable equally to EpCAM.

Identify in the sample whether exist the foranalysis of nucleic acids technology of the polynucleotide of OPN or EpCAM to comprise many technology well-known to those skilled in the art, as Southern engram analysis, Northern engram analysis, dot blot, RNase protection, S1 analysis, amplification technique such as PCR (containing RT-PCR), LCR and in situ hybridization.In the hybridization, for example, target nucleic acid (as the nucleic acid of coding OPN) is discharged the environment in born of the same parents in position, keep cellular form to be used for illustrating and analyzing subsequently (seeing embodiment 1) simultaneously.Following article provides the summary of in situ hybridization: people such as Singer, Biotechniques4:230-250 (1986); People such as Haase, Methods in Virology, vol.VII, pp.189-226 (1984); With NucleicAcid Hybridization:A Practical Approach (people such as Hames compiles .1987).In addition, OPN or EpCAM albumen can detect with aforementioned various immuno analytical method.Specimen usually and positive control (as OPN or the EpCAM that contains reorganization in the sample) and negative control compare.

The present invention also provides the test kit that is used to screen OPN or EpCAM conditioning agent.This test kit can be prepared from very ready-made material and reagent.For example, this test kit comprises one or more of following material: OPN (or EpCAM), test tube, the active specification sheets of detection OPN (or EpCAM).Randomly, test kit can contain the bioactive OPN of tool (or EpCAM).Various test kit and component thereof can prepare according to user's the different needs and the user of specific demand.

The expression of II suppression therapy target spot

For patient HCC, another kind is to suppress the expression of OPN by the means that inhibition OPN activity suppresses the HCC transfer.Equally, can slow down chronic hepatitis patients by inhibition EpCAM expression and develop into HCC.Various different methods well known to those skilled in the art can be used to suppress specifically the expression of specific gene.

The A antisense polynucleotides

Antisense technology has been described the widest, as to be used to realize gene specific inactivation method in scheme, and is the useful tool in research and the diagnosis.For example, the expression that antisense oligonucleotide can high degree of specificity ground suppressor gene, and often illustrate the function of specific gene as the conventional means of bio-science.

The specificity of antisense polynucleotides and susceptibility make it be suitable for the treatment approach.A great number of U.S. patents and science deliver works designed utilize antisense polynucleotides as the treatment animal and human therapeutical agent.Referring to, for example U.S. Patent No. 6,080, and 580; 6,180,403; 6,255,111; 6,306,655; 6,440,739; With 6,524,854.Antisense polynucleotides comprise one with the gene order for the treatment of inactivation (as SEQ ID NO:1 or SEQ ID NO:5) complementary sequence, and length can change, as from less than 10 Nucleotide to greater than 100 Nucleotide, can be applied to object (as the people) safely and effectively.Antisense nucleotide can be Yeast Nucleic Acid oligomerization or poly (RNA) or thymus nucleic acid (DNA) or its stand-in.It can be by bonding (skeleton) between the nucleosides of naturally occurring nuclear base, sugar and covalency, and the oligonucleotide that intimate non-natural exists constitutes.These modifications or the alternate antisense oligonucleotide usually more preferably than natural form because have some advantageous feature, as the affinity of the picked-up, raising and the target nucleic acid that improve cell, and raising is stable in the presence of nuclease.Antisense oligonucleotide of the present invention also can comprise skeleton after the modification or the bonding between non-natural nucleosides.Oligonucleotide skeleton after preferred the modification comprises: thiophosphatephosphorothioate for example, the chirality thiophosphatephosphorothioate, phosphorodithioate, phosphotriester, the aminoalkyl group phosphotriester, the alkyl phosphonate of methylphosphonate and other (comprise 3 '-alkenyl phosphonic acid ester and chiral phosphonate), phosphinate, phosphoramidate (comprise 3 '-phosphoramidic acid acid amides and aminoalkyl group phosphamide), mercapto carbon phosphoramidate, mercapto carbon phosphonate ester, mercapto carbon alkyl phosphotriester, and borine-phosphoric acid ester, they can have normal 3 '-5 ' bonding, 2 '-5 ' similar bonding, and opposite polarity bonding, wherein adjacent nucleosides unit with 3 '-5 ' to 5 '-3 ' or 2 '-5 ' connect to 5 '-2 ' mode.Simultaneously also comprise various salt, mixing salt, and form such as free acid.

In addition, be applicable to that antisense nucleotide of the present invention can be corresponding to encoding sequence and the non-coding sequence of target nucleic acid (as OPN or EpCAM).

B. ribozyme

Use ribozyme can reduce the mRNA level of interested gene (as OPN or EpCAM).Ribozyme is the RNA molecule that enzymic activity is arranged, and can cut or other RNA molecules independently of montage in the special mode of nucleotide sequence.Can be used for ribozyme of the present invention is a kind of RNA molecule with catalysis or enzymic activity, its substrate land and specific RNA target (as the mRNA of OPN or EpCAM) are complementary, and the enzymic activity that has in this target cutting and/or spliced rna, thereby suppress target gene expression.At specific gene and the method for design and use ribozyme is known to those skilled in the art, and in many publications, be described, comprise U.S. Patent No. 6,069,007; 6,107,027; 6,225,291; 6,307,041; 6,482,803; With 6,489,163.

C. little inhibitory RNA (siRNA)

The another kind of useful tool that can reduce purpose mRNA and protein level is little inhibitory RNA (siRNA).The siRNA molecule is little double stranded rna molecule, and it can cause known RNA interfering process, and this is a kind of sequence-specific gene inactivation of form.A kind of RNA interference mechanism hypothesis of proposition, the short dsrna that forms between the antisense strand of mRNA and siRNA can activate the mRNA molecule that depends on ATP and shear.People such as Zamore, Cell101:25-33,2000.Show that RNA disturbs and is present in cells of mamma animals system, ovocyte, body early embryo and some cell type.Referring to for example Elbashir, Sayda M. waits the people, Nature 411:494-497,2001.The siRNA encoding sequence can be based on target-gene sequence (as OPN, or EpCAM) and is designed, and inserts various suitable carrier (as plasmid or virus vector), and these carriers have the transcription initiation and the termination element of correct placement.When being used for required eucaryon acceptor, can use the transcriptional regulatory element of eucaryon.With genetically modified universal method well known to those skilled in the art, the carrier that contains siRNA can be transported to required target.Therefore, RNA disturbs provides another kind of based on sequence and the method for specific inhibition of gene expression, i.e. mRNA by quick degrading genes (as OPN or EpCAM).

D detects the expression of target gene that reduces

Taking curative drug (wherein this medicine contains and can suppress the material that target gene expression (as OPN or EpCAM) is expressed) afterwards, can be by level in the body of target gene before and after relatively taking medicine, the effect of assessment curative drug.The chapters and sections of back are taken elaboration the universal method of pharmaceutical preparation.

When the expression of suppressor gene on transcriptional level when (as reducing the mRNA quantity of target gene), can be by relatively taking the mRNA level of the target gene (as OPN or EpCAM) before and after the curative drug, as adopt Northern engram analysis, dot blot, RT-PCR or the like, thereby confirm that target gene expression descends.The universal method of carrying out this alanysis is well-known to those skilled in the art, and in many documents, be described (see Sambrook and Russell, the same, and people such as Ausubel, the same).

When in the translation skill inhibition of gene expression when (as reducing target gene encoded protein amount), can adopt the means of protein level in the various measurement tissue samples that this area professional knows, relatively take target gene (as OPN or EpCAM) the encoded protein level of curative drug front and back, thereby confirm that target gene expression descends.As previously mentioned, the panimmunity analytical procedure can be used to detect proteins of interest matter (as OPN or EpCAM) routinely existence whether and quantity.At Harlow and Lane, Antibodies, A Laboratory Manual has comprehensive summary to available techniques in 1,988 one books.

The antibody of suitable anti-target protein (as OPN or EpCAM) is that immunoassay is necessary.The universal method for preparing the specific antibody of anti-target protein is well-known to those skilled in the art, and previous section has description.In addition, more existing have the specific antibody of required specificity and be used for immunoassay (as the various monoclonal antibodies of anti-OPN or EpCAM).

In case obtain the specific antibody of target protein (as OPN or EpCAM), patient's target protein level can be measured by various immune analysis method, thereby provides qualitative or quantitative results for the clinician.Patient's various different samples as blood or hepatic tissue, can detect intravital target protein level by the method that immunoassay is described according to more preceding chapters and sections.For immunologic and comprehensive review immune analysis method, can be referring to Stites, the same; U.S. Patent No. 4,366,241; 4,376,110; 4,517,288; With 4,837,168.

V. use and suppress active material of target protein and pharmaceutical composition

Suppress the active material of target protein (as OPN or EpCAM) and can directly be applied to patient, thereby regulate intravital target protein activity.Administering mode can be any common method that is used to make antagonist or inhibitor compound and tissue to be treated finally to contact, for example by tongue or mouthful.Antagonist or inhibitor can be used in any suitable manner randomly with pharmaceutically acceptable carrier.Suitable route of administration such as antagonist or inhibitor is well-known to those skilled in the art, though and a particular composition had multiple route of administration, a certain particular approach usually provides quicker and effective reaction than other approach.

Pharmaceutically acceptable carrier is usually partly determined by particular composition to be administered and the ad hoc approach of using said composition.Therefore, pharmaceutical composition of the present invention can have various appropriate formulation (see RemingtonPharmaceutical Sciences, 17 editions, 1985).

Antagonist or inhibitor individually or with other suitable components, can be made aerosol formulation (be them can by atomization), so that the administration by sucking.Aerosol formulation can place the suitable propelling agent of pressurization, as Refrigerant 12, propane, nitrogen or the like.

The preparation that is fit to administration comprises water-based and non-aqueous solution, isoosmotic sterile solution (they can contain antioxidant, damping fluid, fungistat and be used for providing the solute of isotonicity to preparation), water-based and nonaqueous sterile suspensions (they can contain suspensoid, solubilizing agent, thickening material, stablizer and sanitas).In practical application of the present invention, composition can be by oral, local, intravenously, intraperitoneal, intravesical or intrathecal drug delivery.Randomly, composition also can pass through oral cavity or intranasal administration.Compound formulation may reside in the sealed vessel (as ampoule, bottle) of single dose or multiple doses.Solution and suspension can be by aforesaid sterile powder, granule and tablet preparation.Conditioning agent also can be used as the part of the food for preparing or medicine and administration.

In the context of the present invention, the medication dose that is applied to patient should be enough to produce beneficial effect in for some time in this individuality.This dosage can by signal specific conditioning agent that is adopted and individual state (as the surface-area in body weight or zone to be treated) decision.Dosage size, the generation that also is decided by any unfavorable side reaction that this specific compound or vector administration are followed when the particular individual whether, nature and extent.

When definite patient takes the effective dose of antagonist and inhibitor, the antibody that can assess blood circulation situation, the toxicity of medicine and whether produce anti-this medicine.Usually, for typical individual, the dose equivalent(DE) of antagonist or inhibitor is about 1ng/kg to 10mg/kg.

For administration, antagonist of the present invention and inhibitor administration can be by the speed administrations of measuring, and this speed is the LD of the antagonist when being applied to individual major part and whole body ₅₀Determine with the side reaction of inhibitor under the different concns.Administration can realize by single dose or separate doses mode.

The IV example

Here embodiment that is set forth and example only are used for illustration purpose, various modifications or change form can be prompted to the those skilled in the art that read after the content, thereby these modifications or change form are included among the application's spirit and scope, are also included within the scope of claims.All publications, patent and patent application that this paper mentions all have no restrictedly all to be incorporated herein by reference.

A. embodiment 1: the susceptibility of prediction hepatocellular carcinoma (HCC) diffusion

1. material and method

A) patient and tissue samples

All HCC samples take from Chinese Zhongshan Hospital Attached to Fudan Univ liver cancer research the patient who did excision of informed consent.Is the non-tumour normal liver tissue sample that has obtained 107 couples of primary HCC, transfer HCC and be close to 40 patients of HCC from liver cancer research institute of Chinese Zhongshan Hospital Attached to Fudan Univ (former Shanghai Medical Univ) through hepatectomy and pathological diagnosis.Before the operation, all patients have all done abdominal CT and chest X-ray examination, and part patient has also done the radioisotope scan inspection as required.In 107 pairs of samples, 81 pairs from 27 primary patients HCC, comprise corresponding adjacent non-tumour liver organization and shift HCC[15 there being liver internal diffusion (P group), 12 pairs exist the branch of portal vein TT], 26 pairs from 13 patient and corresponding non-tumour hepatic tissues (not having detectable transfer when performing the operation) thereof that primary HCC is only arranged.Tumour and nonneoplastic tissue are cut into small pieces after excision, and quick-frozen is housed in-70 degree before using in liquid nitrogen.We have confirmed neoplasmic tissue sample with microscope, and its transfer mainly is made of cancer cells, but not do not have the cancer cells of any invasion in the adjacent liver sample of tumour.Among 40 patients, male 39 people, women 1 people.Patient age was from 36 to 74 years old, and intermediate value is 50 years old.The diameter scope of primary HCC is from 1.3-17.5cm, and median diameter is 7.2cm, and wherein 65% (26/40) diameter is greater than 5cm, remaining diameter≤5cm.There is liver cirrhosis in 32 samples (80%).All patient HBV positives except that a people, but nobody HCV positive.Alpha-fetoprotein concentration rising (AFP) in 27 patients (68%) blood (＞20ng/ml).

B) RNA preparation, cDNA dot matrix and hybridization

With reference to the explanation of manufacturers, (Life Technologies Inc.) extracts total RNA in the sample with TRIzol reagent.CDNA microarray (chip) is that american cancer institute advanced techniques center (NCI) makes.Each array contains 9180 cDNA clones, and 7102 genes of having named are wherein arranged, the clone of 1179 EST clones and 122 Incyte.Prepare fluorescently-labeled cDNA with direct labelling method, the hybridization of cDNA microarray is basically according to people such as Wu, Oncogene20:3674-3682, the method described in 2001.In brief, the target material of fluorescence prepares as follows: adopt SuperScript II reversed transcriptive enzyme (Life Technologies), react by oligomerization dT-primer-oligomerization, with Cy3-link coupled thymus nucleic acid (Amersham) mark to total RNA of the non-cancer liver organization of 100 μ g, or with Cy5-link coupled thymus nucleic acid (Amersham) mark to the HCC in 200 μ g former generation or shift on total RNA of tissue.To add in the microarray 42 ℃ of overnight incubation (12-16 hour) after the mixing of target material.Before the hybridization, each microarray with contain 5 * SSC, the prehybridization solution of 0.1%SDS and 1%BSA was 42 ℃ of prehybridizations at least 1 hour.Slide glass is used 2xSSC respectively in room temperature, and 0.1%SDS and 1xSSC and 0.2xSSC respectively wash 2min, wash 1min with 0.05xSSC then.Most samples of mentioning are done double.Each clone's Cy3 and Cy5 fluorescence intensity are rejected background signal with Axon GenePix 4000 scanner collections and with GenePix Pro 3.0 softwares.Filter expression data according to the fluorescence intensity of each passage, some size and flag (flag), calculate the ratio of Cy5/Cy3 then, and in each chip by being that the logarithm-ratio (log-ratio) at center carries out normalized with the intermediate value.

C) data analysis and statistical study

Utilize CLUSTER and TREEVIEW software, adopting with the intermediate value is center correlation method and complete linkage method, carries out unsupervised hierarchical clustering analysis people such as (, the same) Eisen.Analyze for non-supervision and supervision, the BEB-ArrayTools software that we also use ICR biomeasurement research branch office (Biometric Research Branch of the National CancerInstitute) to be developed, this is to be used for carrying out visual and integrated software package statistical study to cDNA dot matrix gene expression data.Use is under the situation of P＜0.001 or 0.002 based on the classification compare tool of single argument F-test in conspicuous level, seeks the gene of differential expression between the predetermined clinical group.Based on the arranged distribution state of the F statistical study of 2000 random alignment, be used to confirm the significance of adding up.During to the diffustivity tumour, in kind use into the t statistics of logarithmic value in more same patient's primary.By using of the arrangement of 2000 P values at random less than 0.001 conspicuous level, according to gene expression atlas, with the compound co-variation forecasting tool of multivariate (Compound Covariate Predictor with the test of " omitting single factor (leave-one-out) " cross validation, CCP), predetermined clinical group is classified.In the step of each cross validation, a sample is omitted, and creates a multivariate CCP based on gene, and wherein said gene is in the training group of being made up of the sample that is not omitted, remarkable monotropic gene under specified level.CCP is used to the sample classification after omitting, and indicates classification then and is correctly or mistake.All to carry out repetition for all samples after one of each eliminating.The mis-classification ratio of total cross validation is determined like this.The mis-classification ratio significance statistically of cross validation determines that by the complete cross validation program that data is repeated 2000 times the member that wherein classifies is a random permutation.CCP is based upon on the weighted linear combination basis of genetic expression variable, and wherein said variable is significantly monotropic in the training group, and its weight is added up corresponding to t-, described in people such as Radmacher (the same).When CCP is used for paired primary and transfer tissue are carried out the branch time-like, also carry out cross validation, wherein omit a pair of data at every turn, and classify based on the paired difference of each genetic expression.The genetic expression average of two repeated sample is used to analyze.

For generation is used for predictive model that the HCC with metastatic potential is classified, we select 10 PN samples and 10 PT samples as a training group at random.The new HCC sample that in test group, comprises 20 double blindings altogether.New sample is classified based on the calculated value of following linear combination: the L=∑ _it _i* (x _i-m _i), t wherein _iThe t value of gene i in the=classification, x _iLogarithm-ratio of gene i in the=new sample to be classified, m _i=gene I is in the intermediate value (seeing Table 2) of PN and PT group.Other details can find in the BRB-ArrayTools user manual.Use comes comparison patient's survival rate based on the WinSTAT software of Excel by the Kaplan-Meier survival analysis.By Cox-Mantel logarithm level test, when PN compares with P or PT, draw and add up the P value.

D). sxemiquantitative PT-PCR and Western trace

Total RNA SUPERSCRIPT ^TMII RNase H-reversed transcriptive enzyme and random hexamer (Invitrogen Inc.) carry out reverse transcription.PCR carried out 26 the circulation (94 ℃, 30sec; 53 ℃, 30sec; 72 ℃ 1min), is 72 ℃ subsequently, an additional cycles of 10 minutes, and adopt following primer: OPN justice 5 '-GACTCGAACGACTCTGATGATGTA-3 ' (SEQ ID NO:3); OPN antisense 5 '-CTGGGCAACGGGGATGG-3 ' (SEQ ID NO:4); And HotStarTaq Master Mix (QLAGEN) test kit.Quantu MRNA ^TM18S (Ambion) is as internal standard.The quantitative employing densitometry of OPN carries out normalization method with the 18S product.The Western engram analysis is basically with reference to people such as Wu, (the same) described method.In brief: with RIPA damping fluid (50mM Tris-HCl, pH7.4/150mM NaCl/1% Triton X-100/1% Deoxycholic Acid/1.0% SDS/1% Trypsin inhibitor,Trasylol) obtains protein lysate from CCL13, SK-Hep-1 and Hep3B cell, separate with 10%SDS-PAGE, be transferred to Immobilin-P film (Millipore, Bedford, MA), with anti--OPN monoclonal antibody (Chemicon International) detection of rat, use mensuration (Amersham) to show then based on ECL.

E) clone and external intrusion analysis

Two kinds of Bel7402 SK-Hep-1 and Hep3B with different metastatic potentials, and the hepatic cell line CCL13 (CCL 13) of a non-convertibility, and be used for determining functional dependency between OPN and the metastatic potential wherein using BD BioCoat according to the explanation of manufacturers ^TMMatrigel ^TMInvade chamber (BD Biosciences) with reference to the description of product.These cells are from American type culture collection.Maintain 37 ℃, 5% CO cell routine ₂Wet environment in, with EMEM (GIBCOL) substratum, and added 10% foetal calf serum, 1 * nonessential amino acid, 1 * Sodium.alpha.-ketopropionate, 2mM L-glutamic acid and penicillin/streptomycin.Analyze for invading, cell is placed in the upper chamber that contains serum-free EMEM, and using mouse OPN (2 μ the g/ml) (R﹠amp that maybe need not recombinate; D Systems) or fully put down in writing neutralizing antibody (3 μ the g/ml) (R﹠amp of anti-OPN; D Systems) under the situation, hatched 20 hours.The EMEM substratum that will contain 5%FBS adds to lower chamber as chemical attractant.Add OPN or OPN antibody before with afterwards, Matrigel is passed through in intrusion ^TMThe cell number of film is counted.

F) histologic analysis

Prepare paraffin-embedded tissue block, be cut into the serial section of 5 micron thickness then, and be laid on the charged slide glass.Slide glass carries out h and E (H﹠amp; E) dyeing.Two pathologists carry out histodiagnosis independently and read sheet.For immunohistochemical analysis, slide glass is taken off cured and carries out immunostaining (pressing people such as Forgues, the described method of J.Biol.Chem.276:22797-22803).In brief, slide recovered antigen in 15 minutes at the 1x citrate buffer solution thereby place in microwave oven, blocked endogenic peroxidase 10 minutes with 3% hydrogen peroxide then.Then with 10% donkey serum sealing non-specific binding, section and mouse-anti OPN antibody (Chemicon International) are in 4 ℃ of overnight incubation.Use the mixture (ABC Elite kit, Vector Labs) of biotinylated two anti-and streptavidin peroxidase.Section is immersed in 3-3 ' diaminobenzidine (DAB) solution (0.25 gram/ml, and contain 3% catalase).Slide dewaters to dimethylbenzene with the Harris-haematoxylin redyeing and with alcohol, with Permount (Sigma) mounting.

2. result

A) the transfevent liver injury primary HCC corresponding with it can't distinguish

For determining the meticulous change in the HCC transfer process, we are with the gene expression atlas of the primary HCC sample of individuality, and with transfer (P group) or portal vein TT (PT group) in the liver that damages with the transfer of mating, promptly P-M or PT-M group compares.Compared the corresponding non-cancer liver organization of each sample simultaneously.At first, we have carried out gene expression atlas relatively [promptly 10 no HCC shift patient's (PN group), 10 PT patients and 10 P patients] to 50 tumor samples with shifting from the original position of 30 patient's stochastic samplings.We attempt to be divided into clinical group with the hierarchical clustering algorithm of non-supervision, these algorithms are composed based on full expression similarity, wherein use 9180 whole genes, perhaps use through the genescreen strainer and getting rid of portion gene (these genes are compared not noticeable change with intermediate value, p＜0.001) about 2487 genes afterwards.Yet this cluster analysis does not produce the significant group result corresponding to clinical grouping.Similarly, we use 107 genes also can't obtain significant grouping, and these genes are compared the gene expression ratio variation and obtained greater than 2 times are filtered with intermediate value.This analytical results means that the HCC of primary and transfer distinguishes by less gene subclass only, and the influence that the cluster analysis of gene may be changed by many other genes, thereby hindered classification.

For studying these fine differences, we have used the classification comparative analysis and the single argument F-test of supervision and have comprehensively arranged test, are defined in the gene of differential expression in the predetermined clinical group.To the analysis of 5 clinical group (being P, P-M, PT, PT-M and PN), 143 remarkable genes (P＜0.005) have been obtained amounting to.Multidimensional scale analysis based on first three main ingredient of 143 remarkable genes discloses, and the PN sample obviously is different from all the other samples, and P, P-M, PT and PT-M sample are that (Fig. 1 a) for undistinguishable.Be that idiopathic gene expression atlas with the transfer HCC tumour of mating can not make a distinction significantly unexpectedly.

B) PN is different from PT and P

In order to confirm and expand above-mentioned discovery that we have carried out the classification comparative analysis to 30 former generation HCC samples (comprising PN, PT and patient P).This analysis has produced the gene of 383 significant differences (P＜0.0005) altogether.Based on these 383 expression of gene spectrums, by the hierarchical clustering algorithm to these 30 PN, P and PT sample classify (Fig. 1 b).Observe two main branches on classification tree, one relevant with the PN sample, and another is relevant with the PT sample with P.P and PT sample do not separate (Fig. 1 b) fully.Therefore, idiopathic nothing shifts the gene expression profile of HCC, obviously is different from the primary HCC that portal vein or elsewhere at the liver soft tissue have the damage shifted.

For further determining that one can accurately be distinguished into the gene set of two predetermine class and in order to differentiate metastasis related gene, we use the machine learning classification algorithm of supervision, it is known multiple correlation prediction procedure (CCP), this algorithm comprises the cross validation test of " omitting single factor ", to avoid excessively estimating this statistical problem of accuracy of prediction, this problem can take place when model is with the same sample training and estimates.This analysis also produces the multivariable prediction value, and this predictor is used for determining that a certain given sample belongs to that class of this two class on earth, has also produced to present the significant list of genes of single argument under given statistical significance level.We use whole gene set and P value＜0.001, will be divided into different pairings according to different clinical criteria from 50 HCC samples of 30 patients, and CCP is used for each pairing (table 1).Under this significance level, the false positive number gene of expecting in classification is less than 10.The mis-classification ratio is confirmed by the cross validation method of " omitting single factor ".For each step (wherein omitting a sample) of cross validation, repeat to select the gene of information and produce the polygene classification.For the probability of the mis-classification ratio that obtains little cross validation at random, can draw by repeating whole cross-validation process, wherein use the class formative of 2000 random alignment to be used for evaluated clinical criteria.Drawn classification P value (table 1) like this.Adopt supervision machine learning classification algorithm, we do not find significant difference (table 1) between PT and PT-M sample.The gene expression atlas of P and PT sample and paired transitivity P-M and PT-M sample be (table 1) much at one.Number gene in these gene classification is in background (false positive) level.These data are consistent with aforementioned cluster and multidimensional scale analysis (multidimensional scaling analysis).

On the contrary, we have predicted tumour (100%) (table 2) exactly with 153 remarkable genes in the sorter from PN and PT sample.The error in classification of cross validation method is much smaller than stochastic prediction (p＜0.005) (table 1).Similarly, we have predicted PN and P sample exactly with a large amount of genes in the sorter, and PN and P/PT sample (table 1).Yet CCP does not produce significantly classification in P, PT, PT-M and P-M, and the number gene in these classification is inapparent.In addition, we find, when tumour size, age, tumour parcel property or liver cirrhosis etc. are used as clinical classification, there is no statistically evident classification.These numerical value are consistent with the result of classification comparative analysis (comprising multidimensional scale analysis and hierarchical clustering Algorithm Analysis).We think that primary and metastatic tumo(u)r have closely similar allelic expression, and the primary HCC tumour of not having a transfer can be different from the primary HCC with portal vein TT or liver internal diffusion.

The performance of table 1. sorter (classifier) in the cross-validation process of " omitting single factor " ^*

The sorter classification **	Clinical grouping	The case sum	The case load of mis-classification	The P value of sorter	Gene number in sorter
						PN vs.PT PN vs.P PN vs.P/PT P vs.PT PT vs.PT-M P/PT vs.P-M/PT-M P vs.PT-M PT vs.P-M tumor size age tumour parcel property cirrhosis	PN PT PN P PN P and PT P PT paired sample paired sample P PT-M PT P-M＞5cm≤5cm＞had or not in 45 years old≤45 years old	????10 ????10 ????10 ????10 ????10 ????20 ????10 ????10 ????10 ????20 ????10 ????10 ????10 ????10 ????16 ????14 ????17 ????13 ????9 ????21 ????14 ????6	????0 ????0 ????1 ????0 ????2 ????0 ????3 ????4 ????3 ????5 ????4 ????3 ????2 ????4 ????7 ????4 ????5 ????7 ????2 ????4 ????7 ????6	??＜0.0005 ??＜0.0005 ??＜0.001 ??0.216 ??0.296 ??0.132 ??0.248 ??0.163 ??0.234 ??0.334 ??0.037 ??0.798	????153 ????157 ????256 ????20 ????1 ????7 ????14 ????9 ????7 ????4 ????13 ????1

^*With composite variable predictor and 9180 gene expression datas altogether, carry out clinical group different classification, the horizontal P=0.001 of significant difference.Sorter is based on 2000 random alignment.The expectation number of false positive gene is 10 in the sorter.

^*PN, single primary HCC; PT has the primary HCC of portal vein TT; PT-M is from the TT of pairing PT; P has the primary HCC of liver internal diffusion; P-M is from the liver internal diffusion of pairing P; P/PT has P and PT simultaneously; P-M/PT-M has P-M and PT-M simultaneously; The tumour size is the diameter of length direction.

C). from the metastatic potential based on measurable patient HCC of model of genetic expression of supervised learning algorithm

Successfully distinguish PN and PT with CCP, make us develop and predict that patient HCC develops into the possibility of transfer based on the model of genetic expression.We select 10 patients PN and 10 patients' PT primary HCC sample to organize as training at random, by predictive model of cross validation classification generation of " omitting single factor ".The classification of training sample produces the tabulation that contains 153 genes.By producing the L value of multiplefactor (multi-factor ial), this provides the fundamentals of forecasting that detects sample, is called as " weighting ballot " exercise (seeing materials and methods).We have comprised that all remaining 20 primary HCC samples are as a test set (15 patients P, 3 extra patients PN and 2 extra patients PT).Fig. 2 has shown calculating " weighting ballot " L value, wherein shifts sample and produces negative value, does not produce positive value and there is the sample of transfer.Except " P " sample (S29), all test sample books all are included into the transfer group, and (Fig. 2 a).Patient's track data shows that patient PN (S56) in operation lung was taken place in back 8 months and shifted, and second patient (S57) be in back 9 months no cancers of operation, and the 3rd patient (S55) do not answer tracking and ask and look into.The set that 153 genes that comparison is obtained based on PN/PT constitute, we have also analyzed these samples by multidimensional scaling.The result shows that S29 expression of gene spectrum more resembles P group and PT group, and does not resemble PN group (Fig. 2 b).This prompting S29 should belong to P group and PT group.Like this, we classify as 18 (90%) among 20 double blinding HCC patients exactly and have metastatic potential.

Table 2 is used for predicting 153 remarkable genes of transfer and calculates the required value of multiplefactor L value at predictive model

UG bunch	Symbol	Describe	The t-value	Intermediate value	The p-value	Unique identification
							?Hs.36566	??LIMK1	Lim domain kinase 1	-7.7122	??-0.433	?0.000000	?160082
?Hs.75573	??CENPE	Kinetochore albumen E (312kD)	-7.2301	???0.217	?0.000001	?160128
							?Hs.81217	??FZD2	Roll up (Drosophila) homologue 2	-7.0334	??-0.499	?0.000002	?160028
?Hs.146580	??ENO2	Hydratase, phosphoenolpyruvate 2, (Y, neuronal)	-6.9978	??-0.238	?0.000002	?160068
							?Hs.222	??ITGA9	Integrin, α 9	-6.699	??-0.159	?0.000004	?160135
?Hs.75887	??COPA	Coatmer albumen mixture, subunit α	-6.4035	??-0.241	?0.000007	?159890
							?Hs.6727	??KIAA0660	Ras-GTP ras GTPase activating protein ras-GTP SH3 structural domain	-6.3742	??-0.281	?0.000007	?160103
?Hs.89578	??GTF2H1	General transcription factor IIH, polypeptide 1	-6.2909	??-0.178	?0.000006	?164987
							?Hs.180941	??VPS41	Letter sorting cavity protein 41 (yeast homologue)	-5.9459	??-0.331	?0.000013	?159888
?Hs.99236	??RGS20	The instrumentality 20 of G-protein signal conduction	-5.8503	??-0.264	?0.000015	?161959
							?Hs.274	??MATK	The Tyrosylprotein kinase that megalokaryocyte is relevant	-5.8166	??-0.366	?0.000016	?160015
?Hs.194816	??STOML1	Probanthine (EBP72) sample albumen 1	-5.7855	??-0.124	?0.000018	?162695
							?Hs.79516	??BASP1	Film adheres to signal protein 1	-5.5974	??-0.415	?0.000026	?159882
?Hs.733	??EPB42	Erythrocyte membrane protein band 4.2	-5.5395	??-0.378	?0.000029	?160067
							?Hs.87539	??ALDH3B2	Aldehyde dehydrogenase 3 families, member B2	-5.5356	??-0.351	?0.000030	?166071

?Hs.5947	????MEL	Mel transforms oncogene	??-5.434	????-0.452	?0.000045	?160104
							?Hs.118354	????CAT56	CAT56 albumen	??-5.4077	????-0.316	?0.000047	?165027
?Hs.27744	????RAB3A	RAB3A, member RAS oncogene family	??-5.35	????-0.338	?0.000044	?160099
							?Hs.7984	????PSCD3	The pleckstrin homologue	??-5.3177	????-0.143	?0.000047	?159887
?Hs.104519	????PLD2	Phospholipase D 2	??-5.2672	????-0.275	?0.000052	?159999
							?Hs.4748	????ADCYAP1R1	Adenylate cyclase activating polypeptide 1	??-5.2037	????-0.166	?0.000060	?161460
?Hs.83155	????ALDH3B1	Aldehyde dehydrogenase 3 families, member B1	??-5.2005	????-0.44	?0.000088	?159838
							?Hs.283822	????RHD	Rhesus blood group, D antigen	??-5.1898	????-0.369	?0.000062	?164821
?Hs.2175	????CSF3R	G CFS 3 acceptors	??-5.1684	????-0.136	?0.000065	?160114
							?Hs.3094	????KIAA0063	The KIAA0063 gene product	??-5.162	????-0.325	?0.000095	?160091
?Hs.119273	????KIAA0296	The KIAA0296 gene product	??-5.132	????-0.545	?0.000070	?159951
							?Hs.23672	????LRP6	LDH receptor related protein 6	??-5.1081	????-1.13	?0.000074	?162040
?Hs.118804	????ENO3	Hydratase, phosphoenolpyruvate 3, (β, muscle)	??-5.0415	????-0.76	?0.000085	?164468
							?Hs.74502	????CTRB1	Chymotrypsinogen B1	??-5.0381	????-0.216	?0.000086	?159787
?Hs.194148	????YES1	V-yes-1 Yamaguchi sarcoma virus oncogene	??-5.0064	????-0.413	?0.000092	?159875
									Unknown (IncytePD:1404153)	??-4.9541	????-0.155	?0.000103	?160122
?Hs.772	????GYS1	Glycogen synthetase 1 (muscle)	??-4.913	????-0.478	?0.000112	?160222
							?Hs.153203	????MDFI	MyoD family inhibition	??-4.8908	????-0.773	?0.000138	?163880
?Hs.247423	????ADD2	Adducin 2 (β)	??-4.8064	????-0.609	?0.000141	?162687
							?Hs.22785	????GABRE	Y-aminobutyric acid (GABA) A acceptor	??-4.8046	????-0.188	?0.000142	?159794
		Unknown (IncytePD:2685601)	??-4.7898	????-0.307	?0.000147	?165108
							?Hs.97087	????CD3Z	CD3Z antigen, zeta polypeptide (TiT3 mixture)	??-4.7723	????-0.487	?0.000152	?160043
?Hs.79006	????DTYMK	Deoxythymidine acid kinase (thymidylate kinase)	??-4.7693	?????0.254	?0.000153	?161858
							?Hs.26915	????SPTBN2	Spectrin, β, non-erythrocytic form 2	??-4.7666	????-0.364	?0.000154	?160846
		Unknown (IncytePD:2509789)	??-4.7523	????-0.175	?0.000159	?164920
							?Hs.38586	????HSD3B1	Hydroxyl-δ-5-steroid dehydrogenase	??-4.7519	????-0.392	?0.000159	?164787
?Hs.32966	????GUCA2B	Guanylate cyclase activator 2B (uroguanyin)	??-4.7519	????-0.368	?0.000159	?164851
							?Hs.12773	????ACOX3	Acetyl-coenzyme A oxydase 3, pristane base (pristanoyl)	??-4.7455	????-0.25	?0.000187	?162487
?Hs.2281	????CHGB	Chromogranin B (secretogranin 1)	??-4.7199	????-0.269	?0.000171	?160078
							?Hs.25197	????STUB1	STIP1 homology and the albumen 1 that contains U-Box	??-4.6897	????-0.264	?0.000183	?160555
?Hs.169536	????RHAG	The glycoprotein that rhesus blood group is relevant	??-4.6648	????-0.326	?0.000193	?164916
							?Hs.96	????PMAIP1	PMA-inducible protein 1	??-4.6573	????-0.124	?0.000196	?160112
?Hs.153053	????CD37	CD37 antigen	??-4.6051	????-0.652	?0.000220	?160033
							?Hs.155227	????EPHB4	????EphB4	??-4.5965	????-0.276	?0.000257	?168938
?Hs.92282	????PITX2	The isostructure domain transcription factor 2 of pairing sample	??-4.584	????-0.149	?0.000230	?160123
							?Hs.79123	????KIAA0084	KIAA0084 albumen	??-4.583	????-0.296	?0.000231	?159886
?Hs.180878	????LPL	Lipoprotein lipase	??-4.5304	????-0.18	?0.000259	?160485
							?Hs.75658	????PYGB	Starch phosphorylase, glycogen; Brain	??-4.5152	?????0.027	?0.000268	?159778
?Hs.286132	????MN7	D15F37 (pseudogene)	??-4.503	????-0.314	?0.000275	?167399
							?Hs.57600	????AP1S1	Adapter associated protein mixture 1	??-4.4656	????-0.26	?0.000299	?160042
?Hs.67688		????EST	??-4.4472	????-0.458	?0.000311	?162920
							?Hs.172458	????IDS	Iduronate 2-sulfatase (Hunt's syndrome)	??-4.4324	????-0.259	?0.000322	?160243
?Hs.80768	????CLCN7	Chloride channel 7	??-4.4298	?????0.058	?0.000324	?161279

?Hs.347527	????SLC20A2	Solute carrier family 20, the member 2	-4.4173	??-0.308	?0.000333	?159936
							?Hs.72550	????HMMR	The mobility acceptor (RHAMM) of hyaluronic mediation	-4.3918	??-0.443	?0.000352	?167575
		Unknown (IncytePD:1681876)	-4.3868	??-0.275	?0.000356	?166536
							?Hs.242947	????DGKI	The diacylglycerol kinases, iota	-4.3835	??-0.369	?0.000358	?161826
?Hs.158249	????KIAA0406	The KIAA0406 gene product	-4.3376	??-0.066	?0.000397	?159825
							?Hs.182577	????INPP5B	Inositol polyphosphate-5-Phosphoric acid esterase, 75kD	-4.315	??-0.269	?0.000417	?160074
?Hs.37054	????EFNA3	????ephrin-A3	-4.3085	??-0.355	?0.000423	?161846
							?Hs.334841	????SELENBP1	Selenium conjugated protein 1	-4.3016	??-0.481	?0.000430	?169315
?Hs.81454	????KHK	Ketohexokinase fructokinase (fructokinase)	-4.2966	??-0.36	?0.000434	?159931
							?Hs.84790	????KIAA0225	KIAA0225 albumen	-4.2732	??-0.151	?0.000582	?160472
?Hs.94498	????LILRA2	Leukocytic immunity sphaeroprotein sample acceptor	-4.2714	??-0.308	?0.000459	?161424
							?Hs.151393	????GCLC	Glutamate-cysteine ligase, catalytic subunit	-4.2523	??-0.421	?0.000479	?166059
?Hs.151738	????MMP9	Matrix metalloproteinase 9	-4.2337	??-0.473	?0.000722	?159912
							?Hs.69707	????HCGII-7	HCGII-7 albumen	-4.2223	???0.802	?0.000512	?161462
?Hs.152251	????FZD5	Roll up (Drosophila) homologue 5	-4.2088	??-0.386	?0.000528	?164899
									Unknown (IncytePD:1570216)	-4.2019	??-0.336	?0.000536	?159962
?Hs.61712	????PDK1	Pyruvic dehydrogenase kinase, isoazyne 1	-4.1746	??-0.251	?0.000570	?160462
							?Hs.66731	????HOXB13	Homology frame (homeo box) B13	-4.1722	??-0.739	?0.000573	?159868
?Hs.80976	????MKI67	The antigen that monoclonal antibody Ki-67 is differentiated	-4.1699	??-0.148	?0.000642	?160039
							?Hs.283664	????ASPH	Aspartic acid beta-hydroxy enzyme	-4.1693	???0.062	?0.000576	?160084
?Hs.76688	????CES1	Procaine esterase 1	-4.1577	??-1.285	?0.000591	?164490
							?Hs.154230	????NDP52	Nuclear structure territory 10 albumen	-4.1483	??-0.178	?0.000604	?159958
?Hs.75596	????IL2RB	The interleukin-22 acceptor, β	-4.1376	??-0.268	?0.000688	?159942
							?Hs.4756	????FEN1	The specific endonuclease 1 of baffle arrangement	-4.1222	???0.195	?0.000640	?160035
?Hs.673	????IL12A	Interleukin 12 A	-4.0844	??-0.082	?0.000696	?162579
							?Hs.89230	????KCNN3	The calcium-activated passage of potassium	-4.0745	???0.008	?0.000711	?161095
?Hs.799	????DTR	The diphtheria toxin acceptor	-4.0616	??-0.421	?0.000812	?167412
							?Hs.120360	????PLA2G6	Phospholipase A2, group VI	-4.0344	??-0.577	?0.000778	?160058
?Hs.171075	????RFC5	Replication factor C (activator 1) 5 (36.5kD)	-4.0263	???0.114	?0.000792	?161332
							?Hs.99899	????TNFSF7	Tumor necrosis factor superfamily, the member 7	-4.0211	??-0.221	?0.000801	?159817
?Hs.9605	????CPSF5	The specific factor 5 of cutting and polyadenylation	-4.0101	???0.079	?0.000821	?159766
							?Hs.95262	????NFRKB	With the conjugated protein relevant nf of kappa B	-4.0081	??-0.162	?0.000825	?167698
?Hs.37129	????SCNN1B	The sodium channel, non-valtage-gated type 1	-4.0053	??-0.244	?0.000830	?161191
							?Hs.296371	????RAB28	RAB28, member RAS oncogene family	-4.0038	???0.343	?0.000833	?160699
?Hs.83795	????IRF2	Interferon regulatory factor 2	-3.9955	??-0.527	?0.000848	?161188
							?Hs.85087	????LTBP4	The TGF-β conjugated protein 4 of hiding	-3.9927	??-0.34	?0.000854	?159923
?Hs.267448	????CGI-85	CGI-85 albumen	-3.986	???0.219	?0.000866	?166502
							?Hs.121521	????ABL2	V-abl murine leukemia virus oncogene homologue 2	-3.9746	??-0.347	?0.000889	?166612
?Hs.28166	????CRSP8	The cofactor of Sp1 transcriptional activation	-3.9714	???0.07	?0.000895	?162996
							?Hs.239706	????GAB1	GRB2 relevant conjugated protein 1	-3.9529	??-0.347	?0.000933	?162416
?Hs.177687	????AKR1C4	Aldehyde-ketone reductase family 1, member C4	-3.9499	???0.145	?0.000939	?161753
							?Hs.25648	????TNFRSF5	The TNF receptor superfamily, the member 5	-3.9371	??-0.147	?0.000966	?166055
?Hs.858	????RELB	V-rel viral oncogene homologue B	-3.935	??-0.12	?0.000971	?164810

Hs.155314	????KIAA0095	The KIAA0095 gene product	?-3.9244	???-0.206	?0.000994	?162213
							Hs.8358	????FLJ20366	The albumen FLJ20366 that supposes	??3.9437	????0.201	?0.000952	?164145
Hs.112819		????EST	??3.9573	????0.217	?0.000924	?168969
							Hs.126263		EST, similar to A38712 scleroproein height	??3.9651	????0.925	?0.000908	?167474
Hs.10669	????DDEF1	Grow differentiation enhancement factor 1	??3.9709	???-0.062	?0.000896	?164026
							Hs.99216		EST is similar to ALU8	??3.9802	????0.288	?0.000878	?169148
Hs.98738	????GRTH	The testis rna helicase enzyme that gonadotropin is regulated	??3.9911	???-0.198	?0.000857	?166657
							Hs.28274		Homo sapiens cDNA:FLJ22049 fis	??3.9912	????0.208	?0.000857	?163989
Hs.186564		????EST	??4.0128	????0.177	?0.000816	?163409
							Hs.34045	????FLJ20764	The albumen FLJ20764 that supposes	??4.0142	????0.325	?0.000814	?168581
Hs.3686	????KIAA0978	KIAA0978 albumen	??4.0211	????0.308	?0.000801	?164187
							Hs.172148		????EST	??4.0307	????0.179	?0.000784	?163746
Hs.239499	????KIAA0185	KIAA0185 albumen	??4.0679	????0.17	?0.000722	?168413
							Hs.169341	????HTPAP	HTPAP albumen	??4.1104	????0.608	?0.000657	?163274
Hs.44131	????KIAA0974	KIAA0974 albumen	??4.1179	????0.828	?0.000646	?164589
							Hs.2969	????SKI	V-ski avian sarcomata virus oncogene homologue	??4.1484	????0.323	?0.000604	?164039
Hs.80618	????FLJ20015	Hypothetical protein	??4.1716	????0.258	?0.000573	?163363
							Hs.136309	????SH3GLB1	The SH3-structural domain, GRB2-sample, the interior film (endophilin) B1	??4.1832	????0.339	?0.000559	?162621
Hs.274293		Homo sapiens mRNA; CDNA DKFZp761G1111	??4.1964	???-0.013	?0.000543	?165504
							Hs.21479	????UBN1	All over nucleoprotein (ubinuclein) 1	??4.2096	????0.554	?0.000527	?167995
Hs.155160	????SRP46	Splicing factor is rich in arginine/Serine, 46kD	??4.2889	????0.291	?0.000442	?168577
							Hs.105584	????RPS6KA4	Ribosomal protein S6 kinases, 90kD, polypeptide 4	??4.3239	????0.349	?0.000409	?168189
Hs.279886	????RANBP9	The RAN bindin 9	??4.336	????0.365	?0.000398	?168730
							Hs.197298	????NS1-BP	NS1-is conjugated protein	??4.346	????0.593	?0.000389	?168257
		Unknown (IncytePD:2895226)	??4.3857	???-0.2	?0.000357	?161881
							Hs.36793	????FLJ23188	The albumen FLJ23188 that supposes	??4.3907	????0.454	?0.000353	?168869
Hs.17384		????EST	??4.3978	???-0.04	?0.000347	?163225
							Hs.78524	????HTCD37	The TcD37 homologue	??4.4097	????0.381	?0.000338	?167570
Hs.2301	????DBH	Dopamine HCL beta-hydroxy enzyme	??4.4196	????0.743	?0.000375	?168202
							Hs.118795	????FLJ10008	The albumen FLJ10008 that supposes	??4.4386	???-0.064	?0.000317	?166653
Hs.33074		The homo sapiens, clone IMAGE:3606519	??4.5036	????0.135	?0.000275	?168589
							Hs.4988		Homo sapiens clone 24711 mRNA sequences	??4.5042	????0.016	?0.000274	?160165
Hs.288872	????FLJ21439	The albumen FLJ21439 that supposes	??4.5242	????0.29	?0.000263	?168393
							Hs.323712	????KIAA0615	The KIAA0615 gene product	??4.5292	????0.024	?0.000260	?163625
Hs.14051		Homo sapiens mRNA; CDNA DKFZp434A2417	??4.5538	????0.215	?0.000246	?168381
							Hs.296287		Be similar to brominated structural domain 4	??4.5576	????0.499	?0.000244	?169290
Hs.57847		EST is similar to the CASPASE-4 precursor	??4.63	????0.264	?0.000208	?165194
							Hs.26289		????EST	??4.7062	????0.948	?0.000176	?169360
Hs.11123	??DKFZP564G092	DKFZP564G092 albumen	??4.9593	????0.476	?0.000101	?163064
							Hs.288908		CDNA:FLJ21913 fis, clone HEP03888	??4.9597	????0.556	?0.000101	?168395
Hs.77495	????UBXD2	Contain UBX structural domain 2	??4.9758	????0.676	?0.000098	?160190
							Hs.24341	????TAZ	What have the PDZ binding motif transcribes the co-activation thing	??5.0014	????0.127	?0.000093	?164176
Hs.50133		????EST	??5.153	????0.243	?0.000067	?168567

?Hs.262958	??DKFZP4348044	The protein D KFZp434B044 that supposes	?5.1851	??0.378	?0.000075	?169042
							?Hs.53478		Homo sapiens cDNA FLJ12366 fis	?5.2202	??0.111	?0.000058	?168383
?Hs.80658	??UCP2	Uncoupling protein 2	?5.2483	??1.308	?0.000054	?168158
							?Hs.209065	??FLJ14225	The albumen FLJ14225 that supposes	?5.3394	??0.468	?0.000045	?164339
?Hs.92357	??GALK1	Lactose kinases 1	?5.6456	??1.15	?0.000037	?169675
							?Hs.50373		????EST	?5.7625	??0.94	?0.000029	?165500
?Hs.266959	??HBG1	Oxyphorase, YA	?5.9704	??1.164	?0.000026	?168326
							?Hs.25566		????EST	?6.1164	??0.182	?0.000009	?168197
?Hs.25277	??FLJ21065	The albumen FLJ21065 that supposes	?6.1957	??0.116	?0.000008	?164202

Above-mentioned predicting the outcome of drawing is divided into two groups with 40 patients, and one is the transfer group, and another is non-transfer group.The Kaplan-Meier survival data is represented, compares with not finding the patient who shifts, and patient's survival time that prediction can be shifted obviously shortens (Fig. 2 C).Because whether the HCC mortality depends on it to a great extent and shift in liver, so our result shows that the gene set that is used for sorter provides the allelic expression accurately of reflection hepatoma Metastasis and survival.

D) osteopontin promotes HCC to shift

Shifting required gene in above-mentioned studies show that, liver should be included in the predictive model.Yet, be based on (P value is 0.001) of strict standard from the tabulation of 153 genes of predictive model so that false-positive gene number in the sorter is minimized, this be correctly classify necessary.So Yan Ge standard may with many for transfer process significant gene foreclose.In order to expand our research, we are to each 10 primary HCC sample in PN group and the PT group, the P value less than 0.002 situation under, the single argument F-that has carried out amounting to 2000 random alignment checks.The false positive that this analysis has produced altogether 224 significance genes and expection is less than 20 (seeing Table 3).In order to identify the gene that causes hepatoma Metastasis, we have detected the tabulation that contains 224 genes, and to mainly in PT and PT-M group, expressing 30 oligogenes that take place significantly to change but in the PN group, seldom change, carried out classifying (seeing Table 4).By using, be that arrange with visual (Fig. 3 a) at the center with these genes by intermediate value by the hierarchical clustering algorithm with the correlation method and the complete linkage method of intermediate value as the center.

In PT group, cross and express average and surpass 3 times but the gene of in PN, not expressing, through being accredited as osteopontin (OPN) (SEQ ID NO:1), osteopontin belongs to the secretor type phosphorprotein, is found it recently and expresses at transfevent mammary cancer, pernicious lung cancer, colorectal carcinoma and prostate cancer camber.Chip expression data comparison result discloses, and in the PT-M sample of a large amount of PT samples and correspondence, the expression of OPN raises, and is extremely low (Fig. 3 b) but express in the PN sample.OPN crosses in the PT sample and expresses, but does not express in the PN sample, and this point has been analyzed by sxemiquantitative RT-PCR and determined (Fig. 3 c and d).To the normal hepatocytes sample of 29 routine primary HCC samples (comprising 16 emerging HCC cases) and 8 healthy organ donors, implement the immunohistochemical analysis (IHC) of OPN.The OPN immune response of these samples is estimated by the double blinding pattern.The tenuigenin OPN dyeing that has the transfevent tumour only is male, especially in highdensity vascular district (Fig. 4).IHC result is basically with chip consistent with the result that RT-PCR obtains (61% positive example, 18 11 of shifting among the HCC) (data not shown).In sum, these studies show that OPN has diagnosis transfevent HCC patient's good value.

For measuring the role of OPN in transfer, we have compared the expression level of OPN in the people HCC clone by Western blotting and the external intrusion analysis of Matrigel.The expression level of OPN is high in SK-Hep-1, is medium in Hep3B, is low (Fig. 5 a), this invasiveness with them conform to (Fig. 5 b) in CCL13.The neutralizing antibody of anti-OPN can be blocked the intrusion (p＜0.04) of SKHep-1 (p＜0.001) and Hep3B cell significantly.Yet the mouse OPN of reorganization does not demonstrate statistically evident hormesis on Hep3B and Sk-Hep-1 cell, and hint or the OPN that tumour cell produced are enough for the phenotype of keeping intrusion, or explanation causes poor efficiency because of difference between species.Also obtained similar result (Fig. 5 c) in 5 extra HCC clones.Yet the existence of neutralizing antibody pair cell and diffusion have only very little influence (Fig. 5 c, right side).

For expanding top discovery, we have measured the effect that OPN shifts to lung the HCC cell in nude mice.The HCCLM3 cell strain is to have a kind of clone that MHCC97 cell that the height lung shifts obtains people such as (, J.Cancer Res.Clin.Oncology, 2002) Li by subcutaneous injection.Week after subcutaneous injection can obtain 100% tumour incidence, and this is with our recent data consistent.Aspect primary tumor big or small, control group and anti-OPN group do not have significant difference (Fig. 5 E), and this is consistent in the external result that can not influence the growth of HCC cell with our anti-OPN antibody.In the 5th week, each mouse in most of I-II level tumours bunch and some III-IV level tumour bunch control groups, all detected lung shift infringement (Fig. 5 E, F).11.1 ± 2.9 tumours bunch are on average arranged in each lung of control group mice.On the contrary, have only only about half of mouse that the lung transfer has taken place in the anti-OPN antibody group, and remaining mouse great majority develop into I level tumour bunch, and 2.6 ± 1.0 tumours bunch are comprehensively on average arranged in each lung, thereby this result is significant (P＜0.01) statistically.Therefore, anti-OPN antibody demonstrates the lung of HCCLM3 cell shifted significant retarding effect.

Table 3. is used for predicting 224 remarkable genes of transfer and calculates the required value of multiplefactor L value at predictive model

UG bunch	Title	Describe	??PN	????PT	The p value	Chromosomal localization	Unique label	The clone
									? Hs.313	OPN	Osteopontin	??1.07	????3.29	??0.00122	????4	??161923	IncytePD：4327691
? Hs.69707	HCGII-7	HCGII-7 albumen	??1.07	????2.85	??0.000512	????6	??161462	IncytePD：1656490
									? Hs.177687	AKR1C4	Aldehyde-ketone reductase family 1, member C4	??0.58	????2.11	??0.000939	????10p15-p14	??161753	IncytePD：5033671
		Unknown	??0.82	????1.74	??0.0018		??161371	IncytePD：3421817
									? Hs.276916	NR1D1	Nuclear receptor subunit family 1, group D, the member 1	??0.74	????1.71	??0.00181	????17q11.2	??166707	IncytePD：1904760
? Hs.211569	GPRK5	G albumen-coupled receptor kinase 5	??0.99	????1.69	??0.00147	????10q24-qter	??161133	IncytePD：1418741
									? Hs.75573	CENPE	Kinetochore albumen E (312kD)	??0.82	????1.65	??1.00E-06	????4q24-q25	??160128	IncytePD：308?1067
? Hs.283664	ASPH	Aspartic acid beta-hydroxy enzyme	??0.7	????1.56	??0.000576	????8q12.1	??160084	IncytePD：3693273
									? Hs.296371	RAB28	RAB28, member RAS oncogene family	??1.07	????1.5	??0.000833	????4p16.1	??160699	IncytePD：1457948
? Hs.89267		EST	??2.49	????1.48	??0.00132	????1	??163570	IncytePD：1633393
									? Hs.79411	RPA2	Replication protein A 2 (32kD)	??1.02	????1.47	??0.00135	????1p35	??167684	IncytePD：1729876
? Hs.79006	DTYMK	Deoxythymidine acid kinase (thymidylate kinase)	??0.98	????1.45	??0.000153	????2	??161858	IncytePD：4818795
									? Hs.26289		EST	??2.59	????1.44	??0.000176	????17	??169360	IncytePD：674211
? Hs.4756	FEN1	The specific endonuclease 1 of baffle arrangement	??0.91	????1.44	??0.00064	????11q12	??160035	IncytePD：2050085
									? Hs.44131	KIAA0974	KIAA0974 albumen	??2.19	????1.44	??0.000646	????10	??164589	IncytePD：4540
? Hs.267448	CGI-85	CGI-85 albumen	??0.96	????1.42	??0.000866	????11q13	??166502	IncytePD：2603232
									? Hs.171075	RFC5	Replication factor C (activator 1) 5 (36.5kD)	??0.83	????1.41	??0.000792	????12q24.2-q24.3	??161332	IncytePD：3590056
? Hs.77495	UBXD2	Contain UBX structural domain 2	??1.88	????1.36	??9.78E-05	????2p14-q21.3	??160190	IncytePD：1940994
									? Hs.184175	C2orf3	Karyomit(e) 2 open reading frame 3	??0.84	????1.36	??0.00139	????2p11.2-p11.1	??166136	IncytePD：2779394
? Hs.146580	ENO2	Hydratase, phosphoenolpyruvate 2, (γ, neuronal 1)	??0.55	????1.31	??1.56E-06	????12p13	??160068	IncytePD：1672630
									? Hs.96	PMAIP1	Phorbol-12-myristic acid-13-acetic ester-inducible protein 1	??0.64	????1.31	??0.000196	????18q22	??160112	IncytePD：1931117
? Hs.80768	CLCN7	Chloride channel 7	??0.83	????1.3	??0.000323	????16p13	??161279	IncytePD：1522646
											Unknown	??0.8	????1.3	??0.00122		??165687	IncytePD：404768
? Hs.9605	CPSF5	The specific factor 5 of cutting and polyadenylation, the 25kD subunit	??0.87	????1.29	??0.000821	????16	??159766	IncytePD：1813371
									? Hs.20295	CHEK1	CHK1 (check point, S.pombe) homologue	??0.85	????1.28	??0.00185	????11q24-q24	??161544	IncytePD：2594058
? Hs.37288	NRID2	Nuclear receptor subunit family 1, group D, the member 2	??0.66	????1.27	??0.00168	????3	??159975	IncytePD：2643094

UG bunch	Title	Describe	??PN	??PT	The p value	Chromosomal localization	Unique label	The clone
									? Hs.75658	PYGB	Starch phosphorylase, glycogen; Brain	??0.83	??1.26	??0.000268	????20p11.2-p11.1	??159778	IncytePD：1975552
? Hs.32058	C1orf19	Karyomit(e) 1 open reading frame 19	??1.85	??1.26	??0.00154	????1q25	??169022	IncytePD：2285569
									? Hs.24994	LOC51098	CGI-53 albumen	??1.75	??1.25	??0.0018	????20	??166179	IncytePD：2347842
? Hs.11123	DKFZP564G0 92	DKFZP564G092 albumen	??1.56	??1.24	??0.000101	????10cen-q26.11	??163064	IncvtePD：2071705
									? Hs.13421	KIAA0056	KIAA0056 albumen	??0.9	??1.24	??0.00119	????11	??159874	IncytePD：1561606
? Hs.28166	CRSP8	The cofactor that the Sp1 transcriptional activation is required, subunit 8 (34kD)	??0.9	??1.23	??0.000895	????5	??162996	IncytePD：1283515
									? Hs.57973	CARD10	Caspase raises domain protein 10	??1.7	??1.23	??0.00197	????22q13.1	??165430	IncytePD：3739467
? Hs.274313	IGFBP6	IGFBP6	??0.87	??1.21	??0.00192	????12q13	??160319	IncytePD：1968126
									? Hs.209065	FLJ14225	The albumen FLJ14225 that supposes	??1.62	??1.18	??4.48E-05	????1q21	??164339	IncytePD：1486385
? Hs.34526	TYMSTR	G albumen-coupled receptor	??0.89	??1.18	??0.00101	????3p21	??161635	IncytePD：2610374
									? Hs.80658	UCP2	Uncoupling protein 2 (mitochondrial, proton carrier)	??5.23	??1.17	??5.44E-05	????11q13	??168158	IncytePD：1907952
? Hs.197298	NS1-BP	NS1-is conjugated protein	??1.95	??1.17	??0.000389	????1q25.1-q31.1	??168257	IncytePD：630045
									? Hs.222	ITGA9	Integrin, α 9	??0.69	??1.16	??3.74E-06	????3p21.3	??160135	IncytePD：2487318
? Hs.288908		Homo sapiens cDNA:FLJ21913 fis, clone HEP03888	??1.87	??1.16	??0.000101		??168395	IncytePD：1938947
									? Hs.21479	UBN1	All over nucleoprotein (ubinuclein) 1	??1.86	??1.16	??0.000527	????16p13.3	??167995	IncytePD：1541201
? Hs.152981	CDS1	CDP-diacylglycerol synthetic enzyme (phosphatidic acid cytidine acyltransferase) 1	??0.81	??1.16	??0.0011	????4q21	??165060	IncytePD：1406071
											Unknown	??0.68	??1.15	??0.000159		??164920	IncytePD：2509789
? Hs.155223	STC2	Tin calsequestrin (stanniocalcin) 2	??0.88	??1.15	??0.00122	????5p14.2-q15	??160310	IncytePD：2823476
									? Hs.1309	CD1A	CD1A antigen, a peptide species	??0.79	??1.15	??0.00161	????1q22-q23	??165058	IncytePD：2906655
? Hs.89230	KCNN3	The calcium activate channel of potassium intermediate/little conduction, subfamily N, the member 3	??0.89	??1.14	??0.000711	????1q21.3	??161095	IncytePD：1747441
									? Hs.331328	FLJ13213	The albumen FLJ13213 that supposes	??0.7	??1.14	??0.00136	????15	??166434	IncytePD：2382190
		Unknown	??0.72	??1.13	??0.000103		??160122	IncytePD：1404153
									? Hs.169341	HTPAP	HTPAP albumen	??2.05	??1.13	??0.000657	????8	??163274	IncytePD：2626340
? Hs.78524	HTCD37	The TcD37 homologue	??1.51	??1.12	??0.000338	????1q21	??167570	IncytePD：1430538
									? Hs.36793	FLJ23188	The albumen FLJ23188 that supposes	??1.68	??1.12	??0.000353	????3p13-q13.33	??168869	IncytePD：2669866
? Hs.154230	NDP52	Nuclear structure territory 10 albumen	??0.7	??1.12	??0.000604	????17q21.3	??159958	IncytePD：1818836

UG bunch	Title	Describe	??PN	??PT	The p value	Chromosomal localization	Unique label	The clone
									? Hs.25648	TNFRSF5	Tumor necrosis factor receptor super family, the member 5	??0.83	??1.12	??0.00132	??20q12-q13.2	??160900	IncytePD：1638346
? Hs.6727	KIAA0660	Ras-GTP ras GTPase activating protein ras-GTP SH3 structural domain-conjugated protein 2	??0.61	??1.11	??6.92E-06	??4q21.1-q21.3	??160103	IncytePD：1899625
									? Hs.8402	ADCY3	Adenylate cyclase	3	??0.77	??1.11	??0.00128	??2p24-p22	??167084	IncytePD：1966824
? Hs.279886	RANBP9	The RAN bindin 9	??1.52	??1.1	??0.000398	??6p23	??168730	IncytePD：1781729
									? Hs.66718	RAD54L	RAD54 (yeast saccharomyces cerevisiae) sample albumen	??0.91	??1.1	??0.00103	??1p32	??166204	IncytePD：2645840
? Hs.10095	LOC56930	The albumen of supposing is from EUROIMAGE 1669387	??1.61	??1.1	??0.00129	??19p13.3	??168579	IncytePD：322585
									? Hs.19348	FLJ13119	The albumen FLJ13119 that supposes	??1.56	??1.1	??0.00131	??15	??169102	IncytePD：1978282
? Hs.194816	STOML1	Stomatin (EBP72) sample albumen 1	??0.77	??1.09	??1.75E-05	??15q24-q25	??162695	IncytePD：1741526
											Unknown	??0.6	??1.09	??0.000147		??165108	IncytePD：2685601
? Hs.84790	KIAA0225	KIAA0225 albumen	??0.75	??1.09	??0.000582	??7	??160472	IncytePD：482519
									? Hs.80976	MKI67	The antigen that monoclonal antibody Ki-67 is differentiated	??0.75	??1.09	??0.000642	??10q25-qter	??160039	IncytePD：2470485
? Hs.89578	GTF2H1	General transcription factor IIH, polypeptide 1 (62kD subunit)	??0.72	??1.08	??6.25E-06	??11p15.1-p14	??164987	IncytePD：37249
									? Hs.27744	RAB3A	RAB3A, member RAS oncogene family	??0.58	??1.08	??4.38E-05	??19p13.2	??160099	IncytePD：1381611
? Hs.2281	CHGB	Chromogranin B (secretogranin 1)	??0.64	??1.08	??0.000171	??20pter-p12	??160078	IncytePD：2821341
									? Hs.92282	PITX2	The isostructure domain transcription factor 2 of pairing sample	??0.75	??1.08	??0.00023	??4q25-q27	??160123	IncytePD：2794019
? Hs.194694	MAP3K6	Mitogen activated protein kinase kinase kinases 6	??0.8	??1.08	??0.00119	??1	??161091	IncytePD：1650939
									? Hs.7984	PSCD3	The pleckstrin homologue, Sec7 and curling/coiled structure territory 3	??0.77	??1.07	??4.69E-05	??7	??159887	IncytePD：3029341
? Hs.158249	KIAA0406	The KIAA0406 gene product	??0.85	??1.07	??0.000397	??20	??159825	IncytePD：1618693
									? Hs.61712	PDK1	Pyruvic dehydrogenase kinase, isoazyne 1	??0.66	??1.07	??0.00057	??2p14-q14.3	??160462	IncytePD：268900
? Hs.126263		EST, similar to A38712 scleroproein height	??3.36	??1.07	??0.000908	??5	??167474	IncytePD：1266194
									? Hs.25648	TNFRSF5	Tumor necrosis factor receptor super family, the member 5	??0.76	??1.07	??0.000966	??20q12-q13.2	??166055	IncytePD：549096
? Hs.239818	PIK3CB	Phosphoinositide-3-kinases, catalytic, beta polypeptides	??0.78	??1.07	??0.00114	??3q24	??160414	IncytePD：267803
									? Hs.656	CDC25C	Cell division cycle protein 25C	??0.79	??1.07	??0.00118	??5q31	??165792	IncytePD：876382
? Hs.288319	SART1	By the squamous cell carcinoma antigen of T cell recognition	??0.62	??1.07	??0.00164	??11cen-q12.3	??164720	IncytePD：2205225
									? Hs.180878	LPL	Lipoprotein lipase	??0.73	??1.06	??0.000259	??8p22	??160485	IncytePD：647128
? Hs.136309	SH3GLB1	The SH3-structural domain, GRB2-sample, the interior film (endophilin) B1	??1.51	??1.06	??0.000559	??1p22	??162621	IncytePD：1552337
									? Hs.3686	KIAA0978	KIAA0978 albumen	??1.44	??1.06	??0.000801	??20	??164187	IncytePD：2234421

UG bunch	Title	Describe	??PN	??PT	The p value	Chromosomal localization	Unique label	The clone
									Hs.146007		Homo sapiens clone IMAGE 21721	??1.75	??1.06	?0.00173	????2	??162822	IncytePD：3143449
Hs.22785	GABRE	Gamma aminobutyric acid (GABA) A acceptor, ε	??0.73	??1.05	?0.000142	????Xq28	??159794	IncytePD：3213034
									Hs.296287		Be similar to brominated structural domain 4, clone IMAGE:3542455	??1.9	??1.05	?0.000244		??169290	IncytePD：2310314
Hs.152251	FZD5	Roll up (Drosophila) homologue 5	??0.56	??1.05	?0.000528	????2q33-q34	??164899	IncytePD：3129290
									Hs.673	IL12A	Interleukin 12 A (natural kill cell stimulating factor 1)	??0.85	??1.05	?0.000696	????3p12-q13.2	??162579	IncytePD：2760318
Hs.155160	SRP46	Splicing factor is rich in arginine/Serine, 46kD	??1.43	??1.04	?0.000442	????11q22	??168577	IncytePD：886075
									Hs.151393	GCLC	Glutamate-cysteine ligase, catalytic subunit	??0.53	??1.04	?0.000479	????6p12	??166059	IncytePD：818192
Hs.82927	AMPD2	Adenosine monophosphate desaminase 2 (isoform L)	??0.82	??1.04	?0.00163	????1p13.3	??162188	IncytePD：1968035
									Hs.2175	CSF3R	G CFS 3 acceptors (granulocyte)	??0.8	??1.03	?6.46E-05	????1p35-p34.3	??160114	IncytePD：1596060
Hs.286132	MN7	D15F37 (pseudogene)	??0.63	??1.03	?0.000275	????15q11-q13	??167399	IncytePD：2739109
									Hs.5716	KIAA0310	The KIAA0310 gene product	??1.39	??1.03	?0.00185	????9q34.2-9q34.3	??169169	IncytePD：1880859
Hs.104519	PLD2	Phospholipase D 2	??0.67	??1.02	?5.23E-05	????17p13.1	??159999	IncytePD：3472725
									Hs.74502	CTRB1	Chymotrypsinogen B1	??0.73	??1.02	?8.55E-05	????16q23-q24.1	??159787	IncytePD：2070278
Hs.288872	FLJ21439	The albumen FLJ21439 that supposes	??1.46	??1.02	?0.000263	????15q14	??168393	IncytePD：1998519
									Hs.57600	AP1S1	Adapter associated protein mixture 1, sigma 1 subunit	??0.69	??1.02	?0.000299	????7	??160042	IncytePD：1804181
Hs.17409	CRIP1	Be rich in the albumen 1 (intestines) of halfcystine	??1.5	??1.02	?0.00123	????7q11.23	??169514	IncytePD：2121863
									Hs.4748	ADCYAP1RI	Adenylate cyclase activating polypeptide 1 (hypophysis) acceptor, I	??0.79	??1.01	?5.99E-05	????7p14	??161460	IncytePD：3214293
Hs.25197	STUB1	STIP1 homology and the albumen 1 that contains U-Box	??0.69	??1.01	?0.000183	????16	??160555	IncytePD：1315677
									Hs.34045	FLJ20764	The albumen FLJ20764 that supposes	??1.56	??1.01	?0.000814	????14	??168581	IncytePD：901577
Hs.95262	NFRKB	With the conjugated protein relevant nf of kappaB	??0.79	??1.01	?0.000825	????11q24-q25	??167698	IncytePD：1685182
									Hs.858	RELB	V-rel fowl Reticuloendotheliosis viral oncogene homologue B (nf of the κ light chain polypeptide genetic enhancer in B-cell 3)	??0.84	??1.01	?0.000971	????19q13.2	??164810	IncytePD：1859449
Hs.180941	VPS41	Letter sorting cavity protein 41 (yeast homologue)	??0.63	??1	?1.26E-05	????7p14-p13	??159888	IncytePD：2910949
									Hs.80618	FLJ20015	Hypothetical protein	??1.43	??1	?0.000573	????17q25	??163363	IncytePD：2043391
Hs.75596	IL2RB	The interleukin-22 acceptor, β	??0.69	??1	?0.000688	????22q13.1	??159942	IncytePD：3936210
									Hs.99216		EST is similar to ALU8_ people Alu subfamily SX sequence	??1.49	??1	?0.000878	????15	??169148	IncytePD：2285350
Hs.155314	KIAA0095	The KIAA0095 gene product	??0.75	??1	?0.000994	????16q22.1-q22.3	??162213	IncytePD：268942

UG bunch	Title	Describe	??PN	????PT	The p value	Chromosomal localization	Unique label	The clone
									? Hs.687	CYP4B1	Cytochrome P450, subfamily IVB, polypeptide 1	??0.85	????1	??0.00114	????1p34-p12	??167183	IncytePD：856900
? Hs.75807	PDLIM1	PDZ and LIM structural domain 1 (elfin)	??1.63	????1	??0.00145	????10q22-q26.3	??160215	IncytePD：2132217
											Unknown	??0.66	????1	??0.00164		??159927	IncytePD：2606307
? Hs.41587	RAD50	RAD50 (yeast saccharomyces cerevisiae) homologue	??0.57	????1	??0.00183	????5q31	??160088	IncytePD：1515426
									? Hs.75887	COPA	Coatmer albumen mixture, subunit α	??0.73	????0.99	??6.55E-06	????1q23-q25	??159890	IncytePD：3296228
? Hs.25566		EST	??1.3	????0.99	??8.89E-06	????22	??168197	IncytePD：948796
									? Hs.274	MATK	The Tyrosylprotein kinase that megalokaryocyte is relevant	??0.61	????0.99	??1.64E-05	????19p13.3	??160015	IncytePD：1515980
? Hs.347527	SLC20A2	Solute carrier family 20 (phosphoric acid translocator), the member 2	??0.66	????0.99	??0.000333	????8p12-q21	??159936	IncytePD：2942938
									? Hs.242947	DGKI	The diacylglycerol kinases, iota	??0.61	????0.99	??0.000358	????7q32.3-q33	??161826	IncytePD：3108609
? Hs.2301	DBH	Dopamine HCL beta-hydroxy enzyme (Dopamine HCL β-monooxygenase)	??2.82	????0.99	??0.000375	????9q34	??168202	IncytePD：1294466
									? Hs.172148		EST	??1.29	????0.99	??0.000784	????5	??163746	IncytePD：929090
? Hs.99899	TNFSF7	Tumour necrosis factor (part) superfamily, the member 7	??0.74	????0.99	??0.000801	????19p13	??159817	IncytePD：2017463
									? Hs.99236	RGS20	The instrumentality 20 of G-protein signal	??0.71	????0.98	??1.53E-05	????8	??161959	IncytePD：4711030
? Hs.262958	DKFZP434B0 44	The protein D KFZp434B044 that supposes	??1.72	????0.98	??7.45E-05	????16	??169042	IncytePD：211389
									? Hs.57847		EST is similar to ICE4_ people CASPASE-4 precursor	??1.47	????0.98	??0.000208	????11	??165194	IncytePD：1362601
? Hs.155227	EPHB4	EphB4	??0.7	????0.98	??0.000257	????7q22	??168938	IncytePD：2056923
									? Hs.72550	HMMR	The mobility acceptor (RHAMM) of hyaluronic mediation	??0.55	????0.98	??0.000352	????5q33.2-qter	??167575	IncytePD：3622417
		Unknown	??0.7	????0.98	??0.000356		??166536	IncytePD：1681876
											Unknown	??0.64	????0.98	??0.000536		??159962	IncytePD：1570216
? Hs.296348	DLST	Dihydrolipoamide S-succsinic acid transferring enzyme	??0.49	????0.98	??0.00151	????14q24.3	??165547	IncytePD：1830335
									? Hs.3094	KIAA0063	The KIAA0063 gene product	??0.66	????0.97	??9.45E-05	????22q13.1	??160091	IncytePD：3227603
? Hs.32966	GUCA2B	Guanylate cyclase activator 2B (uroguanylin)	??0.62	????0.97	??0.000159	????1p34-p33	??164851	IncytePD：1806219
											Unknown	??0.79	????0.97	??0.00121		??164791	IncytePD：3190386
? Hs.190189		EST	??1.33	????0.97	??0.00172	????1	??163286	IncytePD：1679304
									? Hs.733	EPB42	Erythrocyte membrane protein band 4.2	??0.62	????0.96	??2.93E-05	????15q15-q21	??160067	IncytePD：2052032
? Hs.5947	MEL	Mel transforms oncogene-RAB8 homologue	??0.56	????0.96	??4.47E-05	????19p13.1	??160104	IncytePD：1553995

UG bunch	Title	Describe	??PN	??PT	The p value	Chromosomal localization	Unique label	The clone
									Hs.169536	RHAG	The glycoprotein that rhesus blood group is relevant	??0.67	??0.96	??0.000193	????6p21.1-p11	??164916	IncytePD：2048319
Hs.121521	ABL2	Y-abl Abelson murine leukemia virus oncogene homologue 2	??0.64	??0.96	??0.000889	????1q24-q25	??166612	IncytePD：1536149
									Hs.112819		EST	??1.41	??0.96	??0.000924	????1	??168969	IncytePD：2445101
Hs.277445	DGKZ	The diacylglycerol kinases, zeta (104kD)	??0.75	??0.96	??0.00194	????11p11.2	??159822	IncytePD：1875986
									Hs.26915	SPTBN2	Spectrin, β, non-erythrocytic form 2	??0.63	??0.95	??0.000154	????11q13	??160846	IncytePD：1594108
Hs.12773	ACOX3	Acetyl-coenzyme A oxydase 3, pristane base (pristanoyl)	??0.74	??0.95	??0.000187	????4p15.3	??162487	IncytePD：3520054
									Hs.79123	KIAA0084	KIAA0084 albumen	??0.7	??0.95	??0.000231	????3p25.3-p25.1	??159886	IncytePD：2697959
Hs.334841	SELENBP1	Selenium conjugated protein 1	??0.54	??0.95	??0.00043	????1q21-q22	??169315	IncytePD：2591494
									Hs.2969	SKI	V-ski avian sarcomata virus oncogene homologue	??1.65	??0.95	??0.000604	????1q22-q24	??164039	IncytePD：3283271
Hs.37129	SCNN1B	The sodium channel, non-valtage-gated type 1, β (Liddle syndromes)	??0.75	??0.95	??0.00083	????16p12.2-p12.1	??161191	IncytePD：1866654
									Hs.25277	FLJ21065	The albumen FLJ21065 that supposes	??1.25	??0.94	??7.57E-06	????5	??164202	IncytePD：2419078
Hs.83155	ALDH3B1	Aldehyde dehydrogenase 3 families, member B1	??0.58	??0.94	??8.76E-05	????11q13	??159838	IncytePD：2610218
									Hs.24341	TAZ	What have the PDZ binding motif transcribes co-activation thing (TAZ)	??1.27	??0.94	??9.26E-05	????3q23-q24	??164176	IncytePD：2345776
Hs.172458	IDS	Iduronate 2-sulfatase (Hunt's syndrome)	??0.74	??0.94	??0.000322	????Xq28	??160243	IncytePD：549290
									Hs.55279	SERPINB5	Serine (or halfcystine) protease inhibitor, the member 5	??0.62	??0.94	??0.00158	????18q21.3	??162215	IncytePD：460034
Hs.209587		EST is similar to 138022 hypothetical proteins slightly	??1.58	??0.94	??0.00167	????11	??163251	IncytePD：1875433
									Hs.118354	CAT56	CAT56 albumen	??0.69	??0.93	??4.71E-05	????6	??165027	IncytePD：3518549
Hs.182577	INPP5B	Inositol polyphosphate-5-Phosphoric acid esterase, 75kD	??0.74	??0.93	??0.000417	????1p34	??160074	IncytePD：1291948
									Hs.81454	KHK	Ketohexokinase fructokinase (fructokinase)	??0.65	??0.93	??0.000434	????2p23.3-p23.2	??159931	IncytePD：2516508
Hs.76688	CES1	Procaine esterase 1 (monocyte/macrophage Serine lipase 1)	??0.18	??0.93	??0.000591	????16q13-q22.1	??164490	IncytePD：1813269
									Hs.239499	KIAA0185	KIAA0185 albumen	??1.36	??0.93	??0.000722	????10	??168413	IncytePD：514653
Hs.151738	MMP9	Matrix metalloproteinase 9 (gelatinase B, 92kD)	??0.56	??0.93	??0.000722	????20q11.2-q13.1	??159912	IncytePD：1274074
									Hs.186564		EST	??1.38	??0.93	??0.000816	????10	??163409	IncytePD：1640094
Hs.198166	ATF2	Activating transcription factor 2	??0.68	??0.93	??0.00106	????2q32	??160057	IncytePD：2208152
									Hs.149957	RPS6KA1	Ribosomal protein S6 kinases, 90kD, polypeptide 1	??0.75	??0.93	??0.00166	????3	??160006	IncytePD：1822236
Hs.36566	LIMK1	Lim domain kinase 1	??0.6	??0.92	??4.11E-07	????7q11.23	??160082	IncytePD：3373632
									Hs.50133		EST	??1.52	??0.92	??6.67E-05	????4	??168567	IncytePD：1214652

UG bunch	Title	Describe	??PN	??PT	The p value	Chromosomal localization	Unique label	The clone
									? Hs.14051		Homo sapiens mRNA; CDNA DKFZp434A2417	??1.47	??0.92	??0.000246	????10	??168381	IncytePD：1431701
? Hs.66731	HOXB13	Homology frame (homeo box) B13	??0.39	??0.92	??0.000572	????17q21.2	??159868	IncytePD：1861743
									? Hs.85087	LTBP4	Latent transforming growth factor-beta conjugated protein 4	??0.68	??0.92	??0.000854	????19q13.1-q13.2	??159923	IncytePD：1956831
? Hs.239706	GAB1	GRB2 relevant conjugated protein 1	??0.67	??0.92	??0.000933	????4	??162416	IncytePD：5066144
									? Hs.77554		CDNA FLJ14967 fis is similar to zinc finger protein 84	??4	??0.92	??0.0012	????12	??165454	IncytePD：1782052
? Hs.14805	SLC21A11	Solute carrier family 21 (organic anion translocator), the member 11	??1.49	??0.92	??0.00159	????15q26	??168293	IncytePD：408522
									? Hs.94498	LILRA2	Leukocytic immunity sphaeroprotein sample acceptor, subfamily A member 2	??0.71	??0.91	??0.000459	????19q13.4	??161424	IncytePD：3336057
? Hs.799	DTR	The diphtheria toxin acceptor (heparin-in conjunction with the EGF-like growth factor)	??0.61	??0.91	??0.000811	????5q23	??167412	IncytePD：1862257
									? Hs.28274		Homo sapiens cDNA:FLJ22049 fis, clone HEP09444	??1.47	??0.91	??0.000856	????8	??163989	IncytePD：2155690
? Hs.8358	FLJ20366	The albumen FLJ20366 that supposes	??1.46	??0.91	??0.000952	????8p22-q22.3	??164145	IncytePD：3361529
									? Hs.293264		EST	??1.38	??0.91	??0.00107	????11	??168371	IncytePD：829521
? Hs.37953	FANCC	The Fan Keni anaemia, supplementation group C	??0.62	??0.91	??0.00108	????9q22.3	??160036	IncytePD：3669589
									? Hs.250671	FLJ10140	The albumen FLJ10140 that supposes	??1.47	??0.91	??0.00142	????22q13	??168397	IncytePD：642133
? Hs.72964	MKRN3	Makorin, ring finger protein, 3	??0.63	??0.91	??0.00151	????15q11-q13	??164803	IncytePD：3181021
									? Hs.80683	MTRF1	Mitochondrial translation releasing hormone 1	??1.24	??0.91	??0.00161	????13q14.1-14.3	??160533	IncytePD：1462246
? Hs.79516	BASP1	Brain enriches, the signal protein 1 that film adheres to	??0.62	??0.9	??2.60E-05	????5p15.1-p14	??159882	IncytePD：4008301
									? Hs.87539	ALDH3B2	Aldehyde dehydrogenase	3 families, member B2	??0.68	??0.9	??2.96E-05	????11q13	??166071	IncytePD：966447
? Hs.38586	HSD3B1	Hydroxyl-δ-5-steroid dehydrogenase	??0.64	??0.9	??0.000159	????1p13.1	??164787	IncytePD：182802
									? Hs.67688		EST	??0.59	??0.9	??0.000311	????6	??162920	IncytePD：2789893
? Hs.105584	RPS6KA4	Ribosomal protein S6 kinases, 90kD, polypeptide 4	??1.8	??0.9	??0.000409	????11q11-q13	??168189	IncytePD：2110163
									? Hs.24719	MAP-1	Apoptosis regulatory protein 1	??1.45	??0.9	??0.00108	????14q32	??168618	IncytePD：1967338
? Hs.6232	KIAA0764	The KIAA0764 gene product	??1.36	??0.9	??0.00117	????2pter-p25.1	??163561	IncytePD：2043486
									? Hs.73792	CR2	Complement component (3d/Epstein Barr virus) acceptor 2	??0.59	??0.9	??0.00181	????1q32	??160032	IncytePD：3055203
? Hs.134342	LOC55915	At the responsive proteic TASP of the Zorubicin of testis specific	??1.44	??0.9	??0.00185	????7q31.1-7q31.33	??163421	IncytePD：1538396
									? Hs.33074		The homo sapiens, clone IMAGE:3606519, mRNA, part cds	??1.36	??0.89	??0.000275	????8	??168589	IncytePD：1431969
? Hs.83795	IRF2	Interferon regulatory factor 2	??0.54	??0.89	??0.000848	????4q34.1-q35.1	??161188	IncytePD：2174666
									? Hs.81217	FZD2	Roll up (Drosophila) homologue 2	??0.57	??0.88	??1.46E-06	????17q21.1	??160028	IncytePD：2214002

UG bunch	Title	Describe	??PN	??PT	The p value	Chromosomal localization	Unique label	The clone
									? Hs.92357	GALK1	Lactose kinases 1	??5.62	??0.88	??3.65E-05	????17q24	??169675	IncytePD：1215248
? Hs.119273	KIAA0296	The KIAA0296 gene product	??0.53	??0.88	??6.98E-05	????16p13.13- ????16p12.3	??159951	IncytePD：3422646
									? Hs.194148	YES1	V-yes-1 Yamaguchi sarcoma virus oncogene homologue 1	??0.64	??0.88	??9.16E-05	????18p11.31- ????p11.21	??159875	IncytePD：1887736
? Hs.37054	EFNA3	ephrin-A3	??0.69	??0.88	??0.000423	????1q21-q22	??161846	IncytePD：4178495
									? Hs.23643	MST4	Serine/threonine protein kitase MASK	??0.65	??0.88	??0.00108	????X	??163410	IncytePD：2793922
? Hs.266959	HBG1	Oxyphorase, γ A	??5.75	??0.87	??2.57E-05	????11p15.5	??168326	IncytePD：2156647
									? Hs.53478		Homo sapiens cDNA FLJ12366 fis, clone MAMMA1002411	??1.34	??0.87	??5.78E-05	????21	??168383	IncytePD：1366043
? Hs.283822	RHD	Rhesus blood group, D antigen	??0.69	??0.87	??6.17E-05	????1p36.2-p34.1	??164821	IncytePD：1668024
									? Hs.118804	ENO3	Hydratase, phosphoenolpyruvate 3, (β, muscle)	??0.4	??0.87	??8.49E-05	????17pter-p11	??164468	IncytePD：1719955
? Hs.772	GYS1	Glycogen synthetase 1 (muscle)	??0.59	??0.87	??0.000112	????19q13.3	??160222	IncytePD：172916
									? Hs.77448	ALDH4A1	Aldehyde dehydrogenase 4 families, member A1	??0.66	??0.87	??0.00135	????1p36	??166147	IncytePD：831794
? Hs.29640	RECK	Reply inductive, be rich in the albumen of halfcystine, tool kazal motif	??1.42	??0.87	??0.00172	????9p13-p12	??168569	IncytePD：2058483
									? Hs.93780		EST	??1.12	??0.87	??0.00176		??164377	IncytePD：2654539
? Hs.11713	ELF5	E74-like factor 5 (ets structural domain transcription factor)	??0.7	??0.87	??0.0018	????11p13-p15	??161000	IncytePD：2785892
									? Hs.97087	CD3Z	CD3Z antigen, zeta polypeptide (TiT3 mixture)	??0.6	??0.85	??0.000152	????1q22-q23	??160043	IncytePD：3227409
? Hs.118795	FLJ10008	The albumen FLJ10008 that supposes	??1.11	??0.82	??0.000317	????14q22.1-q22.3	??166653	IncytePD：2316425
									? Hs.17384		EST	??1.16	??0.82	??0.000347	????4	??163225	IncytePD：2293931
? Hs.1019	PTHR1	Parathyroid hormone receptor 1	??0.66	??0.82	??0.00102	????3p22-p21.1	??160109	IncytePD：1375235
									? Hs.77667	LY6E	Lymphocyte antigen	6 mixtures, locus E	??0.56	??0.82	??0.00145	????8q24.3	??162145	IncytePD：1472042
? Hs.4988		Homo sapiens clone 24711 mRNA sequences	??1.26	??0.81	??0.000274	????2	??160165	IncytePD：2061405
									? Hs.10669	DDEF1	Grow differentiation enhancement factor 1	??1.14	??0.81	??0.000896	????8q24.1-q24.2	??164026	IncytePD：2507108
? Hs.5353	CASP10	Caspase	10, the L-Cysteine HCL Anhydrous that apoptosis is relevant	??1.01	??0.81	??0.00108	????2q33-q34	??164978										IncytePD：3984879
									? Hs.33102	TFAP2B	Transcription factor AP-1-2 β	??0.58	??0.81	??0.00122	????6p12	??159845	IncytePD：2816550
? Hs.144633	DKFZp434F2 32	The protein D KFZp434F2322 that supposes	??1.22	??0.8	??0.00132	????17q24	??163237	IncytePD：1473265

UG bunch	Title	Describe	??PN	??PT	The p value	Chromosomal localization	Unique label	The clone
									Hs.247423	ADD2	Adducin 2 (β)	??0.55	??0.79	??0.000141	????2p14-p13	??162687	IncytePD：2112288
Hs.323712	KIAA0615	The KIAA0615 gene product	??1.3	??0.79	??0.00026	????16q11.2-q12.2	??163625	IncytePD：1217554
									Hs.120360	PLA2G6	Phospholipase A2, group VI (plasmotype, do not rely on calcium)	??0.57	??0.79	??0.000778	????22q13.1	??160058	IncytePD：1849872
Hs.73800	SELP	Select albumen P (membrane granulosa protein 140kD, antigens c D62)	??0.65	??0.79	??0.00156	????1q22-q25	??160049	IncytePD：3688202
									Hs.65135	KIAA0913	KIAA0913 albumen	??1.16	??0.78	??0.00153	????10	??162465	IncytePD：2752015
		Unknown	??0.99	??0.77	??0.000357		??161881	IncytePD：2895226
									Hs.274293		Homo sapiens mRNA; CDNA DKFZp761G1111	??1.28	??0.77	??0.000542		??165504	IncytePD：530360
Hs.153203	MDFI	MyoD family inhibition	??0.46	??0.75	??0.000138	????6p21	??163880	IncytePD：2645911
									Hs.103393		EST	??1.52	??0.75	??0.0014	????16	??163227	IncytePD：291636
Hs.153053	CD37	CD37 antigen	??0.55	??0.74	??0.00022	????19p13-q13.4	??160033	IncytePD：3041162
									Hs.98738	GRTH	The testis rna helicase enzyme that gonadotropin is regulated	??1.06	??0.72	??0.000857	????11q24	??166657	IncytePD：2404557
Hs.180570	CYP4F12	Cytochrome P450 isoform 4F12	??0.55	??0.72	??0.0014	????19p13.1	??167601	IncytePD：1985566
									Hs.50373		EST	??5.25	??0.7	??2.91E-05	????9	??165500	IncytePD：372922
Hs.131705		EST	??1.01	??0.7	??0.00128	????8	??165368	IncytePD：1921768
									Hs.23672	LRP6	LDH receptor related protein 6	??0.3	??0.69	??7.35E-05	????12p11-p13	??162040	IncytePD：4290851

Table 4. is used for predicting 30 remarkable genes of transfer and calculates the required value of multiplefactor L value at predictive model

UG bunch	Title	Describe	PN	PT	The p value	Chromosomal localization	Unique label	The clone
									? Hs.313	OPN	Osteopontin	1.07	3.29	0.00122	?4	161923	IncytePD：4327691
? Hs.69707	HCGII-7	HCGII-7 albumen	1.07	2.85	0.000512	?6	161462	IncytePD：1656490
									? Hs.177687	AKR1C4	Aldehyde-ketone reductase family 1, member C4	0.58	2.11	0.000939	?10p15-p14	161753	IncytePD：5033671
		Unknown	0.82	1.74	0.0018		161371	IncytePD：3421817
									? Hs.276916	NR1D1	Nuclear receptor subunit family 1, group D, the member 1	0.74	1.71	0.00181	?17q11.2	166707	IncytePD：1904760
? Hs.211569	GPRK5	G albumen-coupled receptor kinase 5	0.99	1.69	0.00147	?10q24-qter	161133	IncytePD：1418741
									? Hs.75573	CENPE	Kinetochore albumen E (312kD)	0.82	1.65	1.00E-06	?4q24-q25	160128	IncytePD：3081067
? Hs.283664	ASPH	Aspartic acid beta-hydroxy enzyme	0.7	1.56	0.000576	?8q12.1	160084	IncytePD：3693273
									? Hs.296371	RAB28	RAB28, member RAS oncogene family	1.07	1.5	0.000833	?4p16.1	160699	IncytePD：1457948
? Hs.274313	IGFBP6	IGFBP6	0.87	1.21	0.00192	?12q13	160319	IncytePD：1968126
									? Hs.34526	TYMSTR	G albumen-coupled receptor	0.89	1.18	0.00101	?3p21	161635	IncytePD：2610374
? Hs.222	ITGA9	Integrin, α 9	0.69	1.16	3.74E-06	?3p21.3	160135	IncytePD：2487318
									? Hs.63984	CDH13	Cadherin 13, the H-cadherin	0.72	1.13	0.000103	?16q24.2- ?q24.3	160122	IncytePD：1404153
? Hs.75596	IL2RB	The interleukin-22 acceptor, β	0.69	1	0.000688	?22q13.1	159942	IncytePD：3936210
									? Hs.55279	SERPINB5	Serine (or halfcystine) protease inhibitor, the member 5	0.62	0.94	0.00158	?18q21.3	162215	IncytePD：460034
? Hs.118354	CAT56	CAT56 albumen	0.69	0.93	4.71E-05	?6	165027	IncytePD：3518549
									? Hs.182577	INPP5B	Inositol polyphosphate-5-Phosphoric acid esterase, 75kD	0.74	0.93	0.000417	?1p34	160074	IncytePD：1291948
? Hs.81454	KHK	Ketohexokinase fructokinase (fructokinase)	0.65	0.93	0.000434	?2p23.3-p23.2	159931	IncytePD：2516508
									? Hs.76688	CES1	Procaine esterase 1 (monocyte/macrophage Serine lipase 1)	0.18	0.93	0.000591	?16q13-q22.1	164490	IncytePD：1813269
? Hs.151738	MMP9	Matrix metalloproteinase 9 (gelatinase B, 92kD)	0.56	0.93	0.000722	?20q11.2- ?q13.1	159912	IncytePD：1274074

UG bunch	Title	Describe	PN	PT	The p value	Chromosomal localization	Unique label	The clone
									Hs.94498	LILRA2	Leukocytic immunity sphaeroprotein sample acceptor, subfamily A member 2	0.71	0.91	?0.000459	19q13.4	161424	IncytePD：3336057
Hs.83795	IRF2	Interferon regulatory factor 2	0.54	0.89	?0.000848	4q34.1-q35.1	161188	IncytePD：2174666
									Hs.81217	FZD2	Roll up (Drosophila) homologue 2	0.57	0.88	?1.46E-06	17q21.1	160028	IncytePD：2214002
Hs.194148	YES1	V-yes-1 Yamaguchi sarcoma virus oncogene homologue 1	0.64	0.88	?9.16E-05	18p11.31- p11.21	159875	IncytePD：1887736
									Hs.23643	MST4	Serine/threonine protein kitase MASK	0.65	0.88	?0.00108	X	163410	IncytePD：2793922
Hs.118804	ENO3	Hydratase, phosphoenolpyruvate 3, (β, muscle)	0.4	0.87	?8.49E-05	17pter-p11	164468	IncytePD：1719955
									Hs.153203	MDFI	MyoD family inhibition	0.46	0.75	?0.000138	6p21	163880	IncytePD：2645911
Hs.153053	CD37	CD37 antigen	0.55	0.74	?0.00022	19p13-q13.4	160033	IncytePD：3041162
									Hs.180570	CYP4F12	Cytochrome P450 isoform 4F12	0.55	0.72	?0.0014	19p13.1	167601	IncytePD：1985566
Hs.23672	LRP6	LDH receptor related protein 6	0.3	0.69	?7.35E-05	12p11-p13	162040	IncytePD：4290851

Embodiment 2: the prediction of easily suffering from hepatocellular carcinoma disease physique

1. materials and methods

A) patient and tissue samples

Prenotice comment portion of Academy of Minnesota University (Institution Review Board) and informed consent, and collect the surgical operation sample according to rules.From 59 suffer from late period chronic hepatic diseases and the patient that between 1995-2001, accepted liver transplantation obtain liver samples.Obtain the normal liver sample in contrast from 8 liver donors.The collection of these samples mainly be by Univ Minnesota-Twin Cities USA's liver organization obtain and distribution system (Liver TissueProcurement and Distribution System LTPADS) carries out.The liver samples of 64 patients' tumour and the non-tumour that is complementary obtains by LTPADS program or Chinese Fudan University liver cancer research.In case obtain freezing sample, organize in the storage frozen at-80 ℃ immediately.

B) cDNA chip

Trizol reagent is adopted in total RNA extracting of freezing sample, and (Invitrogen, Gaithersburg MD) and according to the schedule of operation that manufacturer provides carry out.The spectrophotometry method is adopted in RNA quality control after the extracting, and observes specific 28S and 18S rRNA fragment in the sepharose of 1% concentration.Each part RNA sample equivalent is divided in the pipe, and-80 ℃ of preservations.For the common contrast of cDNA chip, total RNA sample of 8 normal livers is mixed, and all assign in each groove (tub).

Buy the cDNA chip from the NCI advanced techniques center of NIH.These human UniGem v2.0 chips have comprised 9180 cDNA clones, these clones are located in 8281 unique UniGene bunches (based on the Hs Unigene Build#131 that publish February 28 calendar year 2001), EST clone (the Incyte Genomics that has also comprised 122 Incyte, Palo Alto, CA).Hybridizing method adopts the optimizer that NCI sets up (people such as Wu, Oncogene 20:3674-3682,2001; People such as Ye, Nature Med.9:416-423,2003).Adopt GenePix4000 scanner and GenePix Pro software (Axon Instruments, Foster City, CA) acquisition with the fluorescent image behind the chip hybridization.According to recommend about chip test minimum information standard (Minimum Information About a Microarray Experiment Standards) (people such as Brazma A, NatGenet 2001) collected details, can obtain by the Gene Expression Ominibus public database of NCBI.

C) statistical study

Adopt related gene expression ratio (Cy5/Cy3), carry out the dependency that the hierarchical clustering analysis detects phraseology among several list of genes and two risk group.Cluster analysis is adopted Cluster software and is manifested people such as (, the same) Eisen with Tree View software.Be after normalized is carried out at the center, to carry out hierarchical clustering with the intermediate value.

Analyze and adopt BRB ArrayTools to carry out.BRB ArrayTools is that Richard doctor Simon and the Amy Peng by the biological assay of National Cancer Institute research branch sets up.To being from the data measurement on each chip in order to make data normalization and to carry out in the chip relatively.The use of classification contrast instrument is the risk group for relatively more previous definition.The F check is the summary to correlation data t check in two sample packet.With normalized cDNA logarithm ratio, adopt classification contrast instrument to calculate the F check of each gene respectively.The random alignment that this instrument divides into groups.On the basis of these random alignment, the arrangement p value of each gene-correlation in this instrument calculations list.

Come on the basis of analyzing gene expression data in several operational methods of employing, sample is classified into one of two predetermined classifications, and these methods comprise composite variable prediction, the most contiguous K value prediction or supporting carrier machine prediction (support vectormachine predictor).The foundation of this prediction comprises two steps.At first, carry out two sample t checks of standard, be used for identifying and in two groups, express the gene that the ratio logarithmic value has significant difference (reaching 0.001 level).Secondly, the logarithm of the different expressing genes of each sample expression ratio is integrated in the composite variable; This composite variable can be used for the basic classification prediction.The composite variable i of sample i is defined as follows:

$c_i=\underset{j}{\mathrm{\Σ}}{t}_j{x}_{\mathrm{ij}},$

In the formula, tj is that gene j is at two correlated t statistical parameters of group categories.Xij is the logarithm ratio of the gene j that records in sample i, and summation is the gene of expressing at all differences.

We predict the classification of new sample by the linear combination that is calculated as follows:

L＝∑ _it _i*(x _i-m _i)。

Ti is the t value of gene i in the formula, x _iBe the logarithm ratio of gene i in new sample to be classified, m _iBe the mid point value of gene i in two classification.Variable i is included in all significant genes in the original analysis.When L was the positive, new sample should be classified into first phenotypic markers thing, and when L was feminine gender, new sample should be referred to second phenotypic markers thing.

D) expression of EpCAM and vitro inhibition

The expression of EpCAM adopts sxemiquantitative PCR to assess.Total RNA carries out reverse transcription with generation strand cDNA with random primer (Promega), and uses Superscript II ThermoScript II (Invitrogen) according to the schedule of operation of manufacturer.QuantumRNA 18S inherent standard (Ambion) is adopted in the amplification of PCR, uses HotStarTaq archaeal dna polymerase (Qiagen) according to the schedule of operation of manufacturer.The sequence of primer is as follows: forward, 5 '-TGC CGC AGC TCA GGAAGA ATG TGT-3 ' (SEQ ID NO:6); Oppositely, 5 '-CAT CAT TCT GAG TTT TTT GAG AAG-3 ' (SEQ ID NO:7).

Suppress the expression of EpCAM with siRNA.SiRNA is synthetic by Qiagen.The sense strand of EpCAM and antisense strand are: sense strand: 5 '-GUU UGC GGA CUG CAC UUC AdTdT-3 ' (SEQ ID NO:8); Antisense strand: 5 '-ACG UGA CAC GUU CGG AGA AdTdT-3 ' (SEQ ID NO:11).The transfection of siRNA uses TransIT-TKO transfection reagent (Mirus) to carry out according to manufacturer's schedule of operation, and uses the siRNA duplex of 200nm absorbing wavelength in each experiment.The cell growing state is according to the described use Cell Counting Kit-8 of manufacturer (Dojindo MolecularTech.).The experiment triplicate.

2. result

By containing 9128 human cDNA clones' chip, 59 gene expression atlas of suffering from patient's the liver samples that chronic hepatic diseases (CLD) patient and 14 suffer from hepatocellular carcinoma and 8 there is not the gene expression atlas in the normal liver sample of disease compare.Comprise 7 routine viral hepatitis type bs (HBV), 11 routine C type hepatitis (HCV), 3 routine hemochromatosis (HHC), 5 routine WilsonShi diseases (WD), 10 routine alcoholic liver diseases (ALD), the 16 routine sclerosis of primary bile duct (PBC) and 7 routine lupoid hepatitises (AIH) in the CLD sample.With the single argument F checked operation that the random alignment of 2000 group indication things exercises supervision, search the gene of distinguishing these 7 groups of CLD samples.This analysis has obtained amounting to 489 remarkable genes (p＜0.o005).The hierarchical clustering analysis (as descriptions such as Eisen, the same) of 489 genes is shown that these 7 kinds of hepatic diseases groups can be divided into two big classes, and a class is mainly by the composition of sample of HBV, HCV, HHC and WD, the another kind of sample that mainly comprises PBC, ALD and AIH.These results suggest, HBV, HCV, HHC and WD dependency each other are stronger when constituting one group than they and PBC, ALD or AIH.Characterization of molecules by the specificity reflection cause of disease is to the classification results of these samples, and by chance to develop into the risk of liver cell cancer interrelated with them, except the WD sample (data do not show).In order further to determine the difference degree between these groups, by (" leave-one-out ") cross validation and 2000 random alignment tests of carrying out " omitting single factor ", in 7 groups, carry out the composite variable analysis, carry out the t check on this basis.21 simulation tests have been found 500 complex genes altogether.Hierarchical clustering result to these genes is consistent (data do not show) with the result of F check.What conform to it is, PBC, ALD or AIH significantly are different from HBV, HCV, HHC or WD, but the difference between the cause of disease is not clearly (data do not show).This shows that the WD sample belongs to excessive risk group, at least for this set.Explanation to The above results is, distinguishes the gene of low risk group and excessive risk group according to the ability that develops into the liver cell cancer, occupied the characterization of molecules advantage, reflects that the gene of the individual cause of disease is not then preponderated.

In the HBV/HCV/HHC/WD sample jointly imbalance (disregulated) but the gene of not lacking of proper care in ALD/PBC/AIH is assumed to be more relevant with the characterization of molecules of HCC.In order to search this gene set all sidedly, carry out 2000 random alignment tests that the key words sorting thing is tested with (" the leave-one-out ") cross validation of " omitting single factor " with to excessive risk group (HBV/HCV/HHC/WD) and low risk group (ALD/PBC/AIH), the P value less than 0.001 situation under, carried out the most contiguous K value and analyzed (K=3) (3NN) or supporting carrier machine prediction (SVM) computing, this calculative strategy is similar (people such as Ye, the same) to our nearest research.This analysis has produced the combined entry device that comprises 556 remarkable genes, and it gets these two groups fine.It provides significant classification Forecasting Methodology in these groups, adopting the whole accuracy of 3NN method is 78%, adopting the whole accuracy of SVM method is 86%, and cross validation ground mis-classification ratio is starkly lower than desired value (p＜0.0005) (data do not show) at random.Yet, can produce statistically insignificant classification (data do not show) to these sample classifications at random.

Interest be that the many genes in the set that 556 genes constitute can be found in 14 analyzed routine HCC (data not shown goes out).For distinguish in the excessive risk group and 14 routine HCC in the gene of common imbalance, the sample of 14 routine HCC samples and excessive risk group is mixed, adopt 2000 random alignment then, the P value less than 0.001 situation under, compare with 3NN operational method and low risk group.416 genes have been found in this analysis, and wherein 273 genes can be found (49% is overlapping) in the set of 556 genes.These results suggest, the only about half of characterizing gene that can distinguish excessive risk group and low risk group exists in the HCC sample.For the set (table 5) that determines whether 273 genes is the common trait of tumour, we are applied to two independently HCC gene expression atlas to this gene set by 3NN and SVM Forecasting Methodology.A set comprises from 24 HCC samples, and the normal liver identical excessively with above-mentioned use contrast compares; Another set comprises 50 HCC samples, and compares (people such as Ye, the same) with the non-cancer liver organization of its paired.Adopt the SVM method in classification, 273 gene expression characteristicses provide higher fitness, and the whole accuracy of 24 routine HCC samples is that the whole accuracy of 92%, 50 routine HCC sample was 94% (data does not show).Compare with the set of 556 genes, this method has improved overall performance.What conform to it is that the set of 283 non-overlapped genes can not provide any gratifying result.Because most HCC genes involveds have been excluded in non-overlapped genome, the overwhelming majority of 283 genes perhaps belongs to the outer feature of the cause of disease.In addition, 383 overlapping genes choosing from HBV/HCV/HHC/WD and ALD/PBC/AIH/HCC contrast can not draw significant classification for two independent HCC groups, and its whole predicted rate is lower than 50% (random occurrence).273 genes can detect in multiple liver samples, and these samples are from the patient of two HBV with from the diffusion zone liver different piece of 5cm diameter at least.Collection of illustrative plates almost completely identical (data do not show) from 273 genes at the different livers of two patients positions.In addition, preceding 25 genes of parameter p value minimum (p＜0.000001) are to select from the set of 273 genes.This set has produced the result similar to the set of 273 genes (data not shown goes out).Integrate, these results show, the set of 273 genes has comprised most genes that interrelate with HCC, and relevant with the HCC progress, and these genes in ill liver main real be long range diffusion rather than be confined to the original place and keep.

For whether the set that detects 273 genes is the conventional indication thing of human other tumours, utilization SVM method is applied to the gene parameter in this feature from several common chip database datas: and 98 routine liver cell cancers (HCC), 53 routine lung cancer, 89 routine adenocarcinoma of stomach, 37 routine soft tissue neoplasms, 39 routine mammary cancer and 27 routine diffuse large B cell lymphomas (DLBCL) (people such as Alizadeh, the same; People such as Perou, the same; People such as Garber, Proc.NatlAcad.Sci.U.S.A.98:13784-13789,2001).When the set of 273 genes was to 98 extra routine HCC samples performances good (80% sample meets this feature), 97% mammary cancer (39 example) and 78% DLBCL case be feature like the share class also.On the contrary, most from the tumor sample of lung, soft tissue and stomach and the coincidence rate very low (accounting for the 6-30% of all cases) (data not shown goes out) of these features.In contrast, the set of 283 genes (non-HCC associated gene) can not provide gratifying prediction to these samples.Think that thus the HCC associated gene in the sorter is seemingly lacked of proper care jointly in mammary cancer and DLBCL, but in the then not imbalance of adenocarcinoma of lung, soft tissue neoplasm and adenocarcinoma of stomach.

Above-mentioned studies show that, the gene relevant with the HCC morbidity may exist in the set of 273 genes.For example, for expressing the gene that significantly rises and then do not rise, can be used as the oncogene that promotes the cell growth in risk group in the excessive risk group.In order to verify the hypothesis of this " evidence principle " (" proof-of-principle "), we have selected to be positioned in 273 list of genes oligogene at top.This gene is differentiated to be EpCAM, be the relevant calcium ion conducted signal 1 of tumour (TACSTD1, Hs.692), it is expressed in the excessive risk group and has on average improved 3.6 times, but only 1.7 times of (Fig. 6 a), expressions in HCC similar (data not shown goes out) in the low risk group.In excessive risk CLD sample, the high expression level of EpCAM can be proved conclusively (Fig. 6 b) by the quantitative RT-PCR analysis.EpCAM is high expression level in the Hep3B cell, but the expression level in Huh1 and Huh4 cell relatively low (Fig. 6 c), this generally with the growth rate relevant (Fig. 6 d) of cell.In addition, use the special few chain of two different siRNA of EpCAM is suppressed the EpCAM expression, the result causes the growth of Hep3B cell obviously to be suppressed (Fig. 6 f).On the contrary, the few chain of siRNA does not in contrast but have this effect (Fig. 6 e, data not shown goes out).These results show that EpCAM can be by promoting that growth of tumour cell provides carcinogenic nature.

273 remarkable genes, its gene symbol, chromosome map spectral position and UG bunch of mark (knowledge) number are listed in table 5.

Table 5. is used for predicting that chronic hepatitis patients suffers from 273 remarkable genes of HCC possibility and calculate the required value of multiplefactor L value at predictive model

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
										????1	??-7.28	?p＜0.000001	????0.603	????0.903	?160198	Cofilin 2 (muscle)	?? Hs.180141	?? CFL2	????14q
????2	??-6.53	?p＜0.000001	????0.985	????1.607	?168023	The Fc fragment of IgG, high-affinity Ia, acceptor (CD64)	?? Hs.77424	?? FCGR1A	????1q21.2-q21.3
										????3	??-6.46	?p＜0.000001	????0.643	????1.175	?162315	Calcium channel, voltage dependent form, β 3 subunits	?? Hs.250712	?? CACNB3	????12q13
????4	??-6.18	?p＜0.000001	????0.688	????1.112	?160302	Actomyosin IB	?? Hs.121576	?? MYO1B	????2q12-q34
										????5	??-6.16	?p＜0.000001	????0.473	????1.161	?169417	Hemocyanin (ferroxidase)	?? Hs.296634	?? CP	????3q23-q25
????6	??-6.1	?p＜0.000001	????0.876	????1.18	?161756	Albumin	?? Hs.184411	?? ALB	????4q11-q13
										????7	??-6.04	?p＜0.000001	????0.719	????1.224	?162290	UDP-N-acetyl glucosamine pyrophosphorylase 1	?? Hs.21293	?? UAP1	????1q23.1
????8	??-6.01	?p＜0.000001	????0.534	????1.141	?162538	Unknown [homo sapiens], the mRNA sequence	?? Hs.367982		????16
										????9	??-5.94	?p＜0.000001	????0.491	????0.714	?168634	Karyomit(e) 20 open reading frame 3	?? Hs.22391	?? C20orf3	????20p11.22- ????p11.21
????10	??-5.93	?p＜0.000001	????0.756	????1.276	?164136	Acetyl-coa dehydrogenase, long-chain	?? Hs.1209	?? ACADL	????2q34-q35
										????11	??-5.9	?p＜0.000001	????0.864	????1.181	?163874	The KIAA0092 gene product	?? Hs.151791	?? KIAA0092	????11q21
????12	??-5.88	?p＜0.000001	????0.728	????0.925	?163096	CGI-26 albumen	?? Hs.24332	?? CGI-26	????12p12.3
										????13	??-5.73	?p＜0.000001	????0.616	????1.133	?160233	The kinases 3 that dual specific tyrosine-(Y)-phosphorylation is regulated	?? Hs.38018	?? DYRK3	????1q32
????14	??-5.67	?p＜0.000001	????0.786	????1.071	?160436	Be similar to the albumen PRO2831[homo sapiens of supposition], the mRNA sequence	?? Hs.406646		????15
										????15	??-5.65	?p＜0.000001	????0.761	????1.382	?160795	The liver leukemia factor	?? Hs.433707	?? HLF	????17q22
????16	??-5.61	?p＜0.000001	????0.314	????0.798	?161944	Complement component 9	?? Hs.1290	?? C9	????5p14-p12
										????17	??-5.6	?p＜0.000001	????0.506	????0.703	?167718	ATP-binding cassette, subfamily A (ABC1), the member 1	?? Hs.211562	?? ABCA1	????9q31.1
????18	??-5.58	?p＜0.000001	????0.65	????0.912	?168437	KIAA0843 albumen	?? Hs.26777	?? KIAA0843	????5q32
										????19	??-5.57	?p＜0.000001	????0.843	????1.087	?162884	The Phospholipase A2 γ that does not rely on calcium that intracellular membrane is relevant	?? Hs.44198	?? IPLA2(GAMM?? A)	????7q31
????20	??-5.48	?p＜0.000001	????0.657	????1.065	?166910	SIPL albumen	?? Hs.64322	?? SIPL	????2p25.3
										????21	??-5.46	?1.00E-06	????0.544	????1.003	?166192	EST is similar to MT1B_ human metal thioalbumen-IB (MT-1B) [homo sapiens] very much	?? Hs.36102		????16
????22	??-5.46	?1.00E-06	????0.46	????0.832	?164779	N-acetyl-transferase 2 (arylamines N-acetyl-transferase)	?? Hs.2	?? NAT2	????8p22

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
										??23	??-5.44	??1.00E-06	????0.707	????1.191	?166252	CD5 antigen sample albumen (removing the family that acceptor is rich in halfcystine)	?? Hs.52002	?? CD5L	??1q21-q23
??24	??-5.44	??1.00E-06	????0.861	????1.512	?162878	EST is similar to α 1 type XI collagen protein very much, former albumen before the isoform B; Collagen protein XI, α-1 polypeptide [homo sapiens] [H.sapiens]	?? Hs.7967		??1
										??25	??-5.42	??1.00E-06	????0.767	????1.181	?164656	N-chimeric protein (AA 1-299) [homo sapiens], the mRNA sequence	?? Hs.385460		??2
??26	??-5.42	??2.00E-06	????0.803	????1.296	?161780	Incyte?EST	??3441835 ??(IncytePD)
										??27	??-5.38	??1.00E-06	????0.352	????0.745	?160174	Complement component 6	?? Hs.1282	?? C6	??5p13
??28	??-5.35	??2.00E-06	????0.464	????0.875	?160280	Protaminase 2 (blood plasma, carboxypeptidase U)	?? Hs.75572	?? CPB2	??13q14.11
										??29	??-5.34	??2.00E-06	????0.779	????0.978	?163144	KIAA1724 albumen	?? Hs.127243	?? KIAA1724	??2p23.3
??30	??-5.33	??2.00E-06	????0.694	????1.361	?169477	Mannose receptor, C type 1	?? Hs.75182	?? MRC1	??10p13
										??31	??-5.26	??2.00E-06	????0.669	????0.896	?162659	RAB6A, member RAS oncogene family	?? Hs.5636	?? RAB6A	??11q13.3
??32	??-5.25	??2.00E-06	????0.768	????1.052	?161138	Serine (or halfcystine) protease inhibitor, clade A (α-1 protease inhibitor, antitrypsin), the member 1	?? Hs.297681	?? SERPINA1	??14q32.1
										??33	??-5.25	??3.00E-06	????0.685	????1.043	?169635	EST, be similar to slightly extensively transcribe four or three close peptide (tetratricopeptide) duplicate genes, Y chromosome; The TPR gene [homo sapiens] [H.sapiens] of extensively transcribing on the Y chromosome	?? Hs.87980		??2
??34	??-5.21	??3.00E-06	????0.598	????0.815	?162745	Solute carrier family 1 (to neurone/epithelium high-affinity glutamate transporter, system Xag), the member 1	?? Hs.91139	?? SLC1A1	??9p24
										??35	??-5.2	??3.00E-06	????0.371	????0.725	?160366	The proteolytic enzyme 10 of ubiquitin specific	?? Hs.78829	?? USP10	??16q24.1
??36	??-5.16	??3.00E-06	????0.515	????0.932	?166426	Protein S (α)	?? Hs.64016	?? PROS1	??3p11-q11.2
										??38	??-5.14	??4.00E-06	????0.627	????1.044	?162301	The interleukin 1 receptor accessory protein	?? Hs.173880	?? IL1RAP	??3q28
??39	??-5.11	??4.00E-06	????0.534	????0.919	?167159	Steroid-5-5 alpha-reductases, α polypeptide 2 (3-oxo-5 α-steroid δ 4-desaturase α 2)	?? Hs.1989	?? SRD5A2	??2p23
										??40	??-5.04	??5.00E-06	????0.474	????0.9	?167129	Metallothionein(MT) 1L	?? Hs.380778	?? MT1L	??16q13
??41	??-5.02	??5.00E-06	????0.87	????2.237	?163633	The Ieptin acceptor	?? Hs.226627	?? LEPR	??1p31
										??42	??-5.02	??5.00E-06	????0.506	????1.137	?162311	Serine (or halfcystine) protease inhibitor, clade C (antithrombin), the member 1	?? Hs.75599	?? SERPINC1	??1q23-q25.1

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
										??43	??-5.01	??6.00E-06	????0.622	????1.035	?166915	The albumen FLJ12666 that supposes	?? Hs.23767	?? FLJ12666	??1p34.2
??44	??-5	??6.00E-06	????0.741	????1.14	?163572	The protein D KFZp564D0462 that supposes	?? Hs.44197	?? DKFZP564D0?? 46	??6q23.1-q24.3
										??45	??-5	??6.00E-06	????0.842	????1.141	?163676	Inositol (myo)-1 (or 4)-single phosphatase 1	?? Hs.171776	?? IMPA1	??8q21.13-q21.3
??46	??-5	??6.00E-06	????0.903	????1.145	?163549	EST is similar to ARF albumen [homo sapiens] [H.sapiens] slightly	?? Hs.422650		??17
										??47	??-4.99	??6.00E-06	????0.357	????0.608	?168690	Corticotropin-releasing hormone is conjugated protein	?? Hs.115617	?? CRHBP	??5q11.2-q13.3
??48	??-4.99	??6.00E-06	????0.52	????0.846	?169399	Conceived district albumen	?? Hs.74094	?? PZP	??12p13-p12.2
										??49	??-4.98	??6.00E-06	????0.681	????0.994	?162636	Signal recognition particle 54kDa	?? Hs.49346	?? SRP54	??14q13.1
??50	??-4.98	??6.00E-06	????0.633	????0.933	?166021	Inositol polyphosphate-5-Phosphoric acid esterase, 145kDa	?? Hs.155939	?? INPP5D	??2q36-q37
										??51	??-4.93	??7.00E-06	????0.972	????1.427	?159896	Neural precursor is expressed, and grows downward modulation type 4	?? Hs.1565	?? NEDD4	??15q
??52	??-4.92	??8.00E-06	????0.73	????1.087	?163778	N-deacetylase/N-sulfotransferase (heparan glucose amino)) 1	?? Hs.20894	?? NDST1	??5q32-q33.1
										??53	??-4.9	??8.00E-06	????0.705	????1.067	?159807	Kidney contain ankyrin multiple albumen	?? Hs.77546	?? KANK	??9p24.3
??54	??-4.9	??8.00E-06	????0.307	????0.676	?167252	Hydroxy-prostaglandin dehydrogenase 15-(NAD)	?? Hs.77348	?? HPGD	??4q34-q35
										??55	??-4.88	??9.00E-06	????0.724	????1.417	?163254	Lipase A, lysosomal acid, Sterol esterase (WolmanShi disease)	?? Hs.85226	?? LIPA	??10q23.2-q23.3
??56	??-4.87	??1.00E-05	????0.576	????0.923	?162307	The different aspartic acid of protein-L-(D-aspartic acid) O-methyltransgerase	?? Hs.79137	?? PCMT1	??6q24-q25
										??57	??-4.87	??9.00E-06	????0.64	????1.076	?164602	Complement component 1, the s subfraction	?? Hs.169756	?? C1S	??12p13
??58	??-4.83	??1.10E-05	????1.057	????1.872	?164576	Jaw box O1A (rhabdosarcoma)	?? Hs.170133	?? FOXO1A	??13q14.1
										??59	??-4.8	??1.20E-05	????0.78	????1.259	?165739	The gene C G018 that supposes	?? Hs.22174	?? CG018	??13q12-q13
??60	??-4.8	??1.20E-05	????0.719	????1.091	?167087	Solute carrier family 31 (copper transport protein is white), the member 2	?? Hs.24030	?? SLC31A2	??9q31-q32
										??61	??-4.79	??1.20E-05	????0.716	????0.987	?165277	Starch phosphorylase, glycogen; Liver (HersShi disease, glycogen storage diseases type VI)	?? Hs.771	?? PYGL	??14q21-q22
??62	??-4.7	??1.70E-05	????0.766	????1.43	?161801	Solute carrier family 10 (sodium/bile acide cotransport protein family), the member 1	?? Hs.952	?? SLC10A1	??14q24.1
										??63	??-4.7	??1.80E-05	????0.355	????0.917	?162617	FK506 conjugated protein 5	?? Hs.7557	?? FKBP5	??6p21.3-21.2
??64	??-4.68	??1.80E-05	????0.918	????1.294	?163597	The albumen FLJ20366 that supposes	?? Hs.8358	?? FLJ20366	??8q23.2
										??65	??-4.67	??1.90E-05	????0.598	????0.848	?160741	Aldehyde dehydrogenase 8 families, member A1	?? Hs.18443	?? ALDH8A1	??6q23.2
??86	??-4.67	??1.90E-05	????0.392	????0.742	?167158	Complement component 5	?? Hs.1281	?? C5	??9q32-q34
										??67	??-4.65	??2.00E-05	????1.003	????1.661	?165565	Phosphatidylinositols (4,5) bisphosphate 5-Phosphoric acid esterase homologue; Phosphatidyl-4	?? Hs.25156	?? PPI5PIV	??9q34.3

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
																Alcohol Tripyrophosphoric acid 5-Phosphoric acid esterase type IV
??68	??-4.65	??2.00E-05	????0.996	????1.169	??160476	It may be the mouse homologue of disappearance in polyposis 1	?? Hs.178112	???? DP1	?5q22-q23
										??69	??-4.65	??2.50E-05	????0.93	????1.204	??161778	Protein phosphatase 1 D magnesium dependent form, the δ isoform	?? Hs.100980	???? PPM1D	?17q23.2
??70	??-4.62	??2.20E-05	????0.875	????1.013	??164997	N-acetylgalactosaminase, α-	?? Hs.75372	???? NAGA	?22q13-qter
										??71	??-4.62	??2.30E-05	????1.04	????1.351	??160731	Histone deacetylase 6	?? Hs.6764	???? HDAC6	?Xp11.23
??72	??-4.62	??2.30E-05	????0.98	????1.326	??168995	Ring finger protein 13	?? Hs.6900	???? RNF13	?3q25.1
										??73	??-4.6	??2.40E-05	????0.536	????0.805	??163500	Plasma thromboplastin antecedent (plasma thromboplastin antecedent)	?? Hs.1430	???? F11	?4q35
??74	??-4.59	??2.50E-05	????0.359	????0.544	??159810	C-type lectin BIMLEC precursor	?? Hs.2441	???? BIMLEC	?2q24.2
										??75	??-4.57	??2.60E-05	????0.912	????1.66	??168655	Complement component 1, q subfraction, beta polypeptides	?? Hs.8986	???? C1QB	?1p36.3-p34.1
??76	??-4.57	??2.70E-05	????0.529	????1.031	??166497	Histadine ammoniacal	?? Hs.276590	???? HAL	?12q22-q24.1
										??77	??-4.57	??3.60E-05	????0.421	????0.88	??161748	Acetyl-CoA Transacetylase 1 (acetic acid acetyl-CoA thiolase)	?? Hs.37	???? ACAT1	?11q22.3-q23.1
??78	??-4.56	??2.70E-05	????0.636	????1.205	??164394	CD163 antigen	?? Hs.74076	???? CD163	?12p13.3
										??79	??-4.54	??2.90E-05	????0.926	????1.178	??160011	General transcription factor IIA, 2,12kDa	?? Hs.76362	???? GTF2A2	?15q21.3
??80	??-4.54	??3.10E-05	????0.634	????0.922	??161895	Nuclear receptor subunit family 1, group I, the member 2	?? Hs.118138	???? NR1I2	?3q12-q13.3
										??81	??-4.54	??3.00E-05	????0.907	????1.181	??167754	Homo sapiens mRNA total length is inserted cDNA clone EUROIMAGE 926491, mRNA sequence	?? Hs.98401		?19
??82	??-4.54	??4.10E-05	????0.988	????1.3	??161838	Nadh dehydrogenase (ubiquinone) 1, inferior mixture, the unknown, 1,6kDa	?? Hs.84549	???? NDUFC1	?4q28.2-q31.1
										??83	??-4.47	??3.70E-05	????1.124	????1.642	??161856	Glutathione-S-transferase sample albumen; Thiadiazolidine isomerase omega	?? Hs.11465	???? GSTTLp28	?10q24.33
??84	??-4.47	??3.80E-05	????0.893	????1.216	??163456	Phytane base-CoA hydroxylase (RefsumShi disease)	?? Hs.172887	???? PHYH	?10pter-p11.2
										??85	??-4.46	??3.90E-05	????0.51	????0.865	??168256	The B-factor, properdin	?? Hs.69771	???? BF	?6p21.3
??86	??-4.43	??4.30E-05	????0.611	????1.011	??162472	Angiogenin, rnase, RNase A family, 5	?? Hs.332764	???? ANG	?14q11.1-q11.2
										??87	??-4.41	??4.80E-05	????0.593	????0.906	??167629	N-acetyl-transferase 1 (arylamines N-acetyl-transferase)	?? Hs.155956	???? NAT1	?8p23.1-p21.3
??88	??-4.39	??5.90E-05	????0.884	????1.231	??162036	The Dombrock blood group	?? Hs.13776	???? DO	?12q13.2-q13.3
										??90	??-4.39	??5.00E-05	????0.448	????0.831	??159972	PBEF	?? Hs.239138	???? PBEF	?7q22.1
??91	??-4.38	??5.10E-05	????0.892	????1.14	??160759	Glucuronidase, β	?? Hs.183868	???? GUSB	?7q21.11
										??92	??-4.37	??5.20E-05	????0.797	????1.284	??162192	Acetyl-coa dehydrogenase, C-4 to C-12 straight chain	?? Hs.79158	???? ACADM	?1p31

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
										??93	??-4.37	??5.40E-05	????0.811	????1.062	?161636	Homo sapiens clone 24405 mRNA sequences	?? Hs.23729		????1
??94	??-4.34	??5.80E-05	????0.746	????1.211	?168452	Methene tetrahydrofolate dehydrogenase (NADP+ dependent form), anhydroleucovorin cyclohydrolase, Tetrahydrofolic formylase	?? Hs.172665	???? MTHFD1	????14q24
										??95	??-4.33	??6.10E-05	????0.541	????0.906	?165666	Rnase, RNase A family, 4	?? Hs.283749	???? RNASE4	????14q11.1
??96	??-4.33	??6.20E-05	????0.482	????0.939	?167394	Butyrylcholine esterase	?? Hs.1327	???? BCHE	????3q26.1-q26.2
										??97	??-4.3	??6.80E-05	????0.62	????0.767	?167501	Propionyl CoA carboxylase, the α polypeptide	?? Hs.80741	???? PCCA	????13q32
??98	??-4.3	??6.80E-05	????0.809	????2.181	?165974	Insulin-like growth factor binding protein 1	?? Hs.102122	???? IGFBP1	????7p13-p12
										??99	??-4.29	??7.00E-05	????0.622	????0.933	?161234	Have a liking for spot albumen (plakophilin) 2	?? Hs.25051	???? PKP2	????12p11
??100	??-4.29	??7.00E-05	????0.852	????1.098	?166532	The Yelkin TTS transfer protein	?? Hs.285218	???? PCTP	????17q21-q24
										??101	??-4.28	??7.40E-05	????0.567	????0.815	?167750	E.C. 2.7.1.20	?? Hs.432422	???? ADK	????10cen-q24
??102	??-4.27	??7.80E-05	????0.479	????0.766	?165890	Fibrinogen, the B beta polypeptides	?? Hs.7645	???? FGB	????4q28
										??103	??-4.26	??7.70E-05	????0.406	????0.89	?161362	Tryptophane 2,3-dioxygenase enzyme	?? Hs.183671	???? TDO2	????4q31-q32
??104	??-4.25	??8.00E-05	????0.739	????1.044	?159764	Annexin A7	?? Hs.386741	???? ANXA7	????10q21.1-q21.2
										??105	??-4.25	??8.10E-05	????0.642	????0.88	?164249	Aminocarboxymuconate-semialdehyde decarboxylase	?? Hs.114088	???? ACMSD	????2q21.2
??106	??-4.24	??8.30E-05	????0.91	????1.142	?162711	Split and melt element (mitofusin) 2	?? Hs.3363	???? MFN2	????1p36.21
										??107	??-4.24	??8.30E-05	????0.784	????1.391	?160370	The kinases that serum/glucocorticosteroid is regulated	?? Hs.296323	???? SGK	????6q23
??108	??-4.24	??8.40E-05	????0.483	????0.867	?161146	3-hydroxy steroid epimerase	?? Hs.11958	???? RODH	????12q13
										??109	??-4.23	??9.10E-05	????0.476	????0.846	?161986	Tumor rejection antigen (gp96) 1	?? Hs.82689	???? TRA1	????12q24.2-q24.3
??110	??-4.23	??8.60E-05	????0.807	????1.049	?165670	Toll sample acceptor 2	?? Hs.63668	???? TLR2	????4q32
										??111	??-4.22	??8.80E-05	????0.577	????0.78	?166820	The KIAA0212 gene product	?? Hs.154332	???? KIAA0212	????3p26.1
??112	??-4.21	??9.10E-05	????0.604	????0.838	?164495	The homo sapiens, clone IMAGE:3833472, mRNA, mRNA sequence	?? Hs.234898		????12
										??113	??-4.21	??9.10E-05	????0.407	????0.592	?163893	Scleroproein former state albumen 1	?? Hs.107	???? FGL1	????8p22-p21.3
??114	??-4.2	??9.30E-05	????0.651	????1.058	?167287	Cytochrome b-5	?? Hs.83834	???? CYB5	????18q23
										??115	??-4.2	??9.40E-05	????0.597	????1.015	?162446	The flavoprotein dehydrogenase of metastatic electron	?? Hs.323468	???? ETFDH	????4q32-q35
??116	??-4.19	??9.90E-05	????0.507	????1.102	?169375	Cytochrome P450, subfamily IIC (mephenytoin 4-hydroxylase), polypeptide 9	?? Hs.167529	???? CYP2C9	????10q24
										??117	??-4.18	??0.000103	????0.523	????0.963	?160720	SODH	?? Hs.878	???? SORD	????15q15.3

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
										?118	-4.17	?0.000107	????0.992	????1.266	?162067	Splicing factor 3b, subunit 1,155kDa	?? Hs.334826	?? SF3B1	????2q33.1
?119	-4.15	?0.000115	????0.639	????0.936	?164393	Homo sapiens mRNA; CDNA DKFZp762O1615 (from clone DKFZp762O1615), the mRNA sequence	?? Hs.284252		????5
										?120	-4.15	?0.000114	????0.794	????1.029	?162329	The antigen that the estrogen receptor binding site is relevant, 9	?? Hs.9222	?? EBAG9	????8q23
?121	-4.14	?0.000116	????0.59	????1.176	?164863	Solute carrier family 2 (auxiliary glucose transporter), the member 2	?? Hs.167584	?? SLC2A2	????3q26.1-q26.2
										?122	-4.14	?0.000117	????0.767	????1.029	?163052	The toes homologue (mouse) that merges	?? Hs.288929	?? FTS	????16q12.1
?123	-4.12	?0.000124	????0.712	????0.997	?160399	cullin?3	?? Hs.78946	?? CUL3	????2q36.3
										?124	-4.12	?0.000124	????0.649	????0.837	?165894	Protein kinase, cAMP dependent form, regulation and control, II type, β	?? Hs.77439	?? PRKAR2B	????7q22-q31.1
?125	-4.11	?0.000126	????0.941	????1.258	?162938	PTD013 albumen	?? Hs.22679	?? PTD013	????6q13-q22.33
										?126	-4.09	?0.000137	????0.622	????0.958	?160328	Preceding α (sphaeroprotein) inhibition, the H3 polypeptide	?? Hs.76716	?? ITIH3	????3p21.2-p21.1
?127	-4.08	?0.000142	????0.718	????1.057	?165794	Epoxide hydrolase 2, cytoplasm type	?? Hs.113	?? EPHX2	????8p21-p12
										?128	-4.07	?0.000149	????0.405	????0.709	?162561	Rna helicase enzyme associated protein [homo sapiens], the mRNA sequence	?? Hs.381097		????16
?129	-4.06	?0.000149	????0.447	????0.743	?168811	Acetyl-CoA Transacetylase 1 (acetate acetyl-CoA thiolase)	?? Hs.37	?? ACAT1	????11q22.3-q23.1
										?130	-4.06	?0.000152	????0.949	????1.293	?169563	Zinc finger protein 10 3 homologues (mouse)	?? Hs.155968	?? ZFP103	????2p11.2
?131	-4.05	?0.000155	????0.565	????1.142	?162666	Prokinin	?? Hs.77741	?? KNG	????3q27
										?132	-4.05	?0.000156	????0.353	????0.729	?168282	Group-specific component (vitamin D binding protein)	?? Hs.198246	?? GC	????4q12-q13
?133	-4.05	?0.000157	????0.678	????0.841	?168476	The plain 88kDa of nucleopore	?? Hs.172108	?? NUP88	????17p13.2
										?134	-4.04	?0.000161	????0.66	????1.011	?167801	Sec23 homologue A (yeast saccharomyces cerevisiae)	?? Hs.272927	?? SEC23A	????14q13.2
?135	-4.01	?0.00018	????0.624	????0.786	?165731	Oncoprotein D52 sample albumen 1	?? Hs.16611	?? TPD52L1	????6q22-q23
										?136	-4.01	?0.000177	????0.586	????0.97	?169253	Paraoxonase 3	?? Hs.335322	?? PON3	????7q21.3
?137	-4.01	?0.000179	????0.841	????1.036	?159850	Homo sapiens cDNA FLJ34315 fis, clone FEBRA2008341, mRNA sequence	?? Hs.376655		????14
										?138	-4	?0.000182	????0.69	????1.057	?167281	Cell division cycle protein 2-sample albumen 5 (Pseudocholinesterase relevant cell division control thing)	?? Hs.59498	?? CDC2L5	????7p13
?139	-4	?0.000185	????0.589	????0.913	?165590	Translocator 1	?? Hs.8146	?? TLOC1	????3q26.2-q27
										?140	-3.99	?0.00019	????0.69	????0.939	?162599	Haptoglobin	?? Hs.75990	?? HP	????16q22.1
?141	-3.97	?0.000202	????0.79	????0.997	?164028	EST is similar to ATDA_ people's diamino Transacetylase (spermidine/essence slightly	?? Hs.356269		????X

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
																Amine N (1)-Transacetylase) (SSAT) (putrescine acetyltransferase) [H.sapiens]
?142	-3.97	??0.000205	????0.418	????0.896	?166007	Tyrosine aminotransferase	?? Hs.161640	?? TAT	????16q22.1
										?143	-3.95	??0.000219	????0.828	????1.188	?165559	C-mer proto-oncogene Tyrosylprotein kinase	?? Hs.306178	?? MERTK	????2q14.1
?144	-3.95	??0.000221	????0.816	????1.224	?165133	Basic leucine zipper and W2 structural domain 1	?? Hs.155291	?? BZW1	????2q33
										?145	-3.94	??0.000223	????0.334	????0.522	?167542	KIAA0062 albumen	?? Hs.89868	?? KIAA0062	????8p21.2
?146	-3.93	??0.00023	????0.504	????0.902	?169449	Arginase, liver	??166337 ??(1ncytePD)
										?147	-3.93	??0.000231	????0.649	????0.78	?167543	Blood coagulation factor VIII, former setting accelerator component (hemophilia A)	?? Hs.79345	?? F8	????Xq28
?148	-3.93	??0.000235	????0.491	????0.61	?163368	CDw92 antigen	?? Hs.179902	?? CDW92	????9q31.2
										?149	-3.91	??0.000244	????1.059	????1.761	?168931	Heat shock protein(HSP) 105kD	?? Hs.36927	?? HSP105B	????13q12.2
?150	-3.91	??0.000245	????0.406	????0.687	?165009	Orosomucoid 1	?? Hs.572	?? ORM1	????9q31-q32
										?151	-3.89	??0.000264	????0.37	????0.662	?162162	Complement component 8, the α polypeptide	?? Hs.93210	?? C8A	????1p32
?152	-3.89	??0.000265	????0.746	????1.159	?166110	2,4-diene acyl CoA reductase enzyme 1, mitochondrial	?? Hs.81548	?? DECR1	????8q21.3
										?153	-3.88	??0.000277	????0.749	????0.985	?161689	Growth hormone receptor	?? Hs.125180	?? GHR	????5p13-p12
?154	-3.87	??0.000282	????0.899	????1.223	?167617	Seleno-protein P, blood plasma, 1	?? Hs.275775	?? SEPP1	????5q31
										?155	-3.86	??0.000291	????0.644	????0.938	?161484	Cytochrome P450, subfamily IVF, polypeptide 3 (leukotriene B4 omega hydroxylase)	?? Hs.106242	?? CYP4F3	????19p13.2
?156	-3.85	??0.000298	????0.91	????1.172	?167551	The albumen 7 that microtubule is relevant	?? Hs.146388	?? MAP7	????6q23.2
										?157	-3.85	??0.000299	????0.604	????0.895	?169703	Glucophosphomutase 1	?? Hs.1869	?? PGM1	????1p31
?158	-3.85	??0.000305	????0.673	????0.909	?163040	Incyte?EST	??2593385 ??(IncytePD)
										?159	-3.84	??0.000311	????0.602	????0.807	?165566	L-3-glycoloyl-coa dehydrogenase, short chain	??1550727 ??(IncytePD)
?160	-3.83	??0.000322	????1.017	????1.192	?162707	Homo sapiens clone 25038mRNA sequence	?? Hs.306359		????15
										?161	-3.83	??0.000322	????0.423	????0.652	?166674	Paired primary amino acid diced system 4	?? Hs.170414	?? PACE4	????15q26
?162	-3.82	??0.000327	????0.732	????1.378	?165737	Fatty acid binding protein 1, liver	?? Hs.380135	?? FABP1	????2p11

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
										?163	-3.82	?0.000334	????0.596	????0.86	?168366	Sterol carrier protein 2	?? Hs.75760	???? SCP2	????1p32
?164	-3.82	?0.000334	????0.809	????1.044	?165115	Aconitase 1, solvable type	?? Hs.154721	???? ACO1	????9p22-p13
										?165	-3.82	?0.000389	????0.718	????1.152	?161732	plexin?B1	?? Hs.278311	???? PLXNB1	????3p21.31
?166	-3.8	?0.000349	????0.854	????1.28	?162202	Transferrins,iron complexes	?? Hs.396489	???? TF	????3q21
										?167	-3.79	?0.000361	????0.553	????0.886	?167991	Hydroxy steroid (17-β) desaturase 4	?? Hs.75441	???? HSD17B4	????5q21
?168	-3.79	?0.000365	????0.662	????0.953	?169717	PgR membrane component 1	?? Hs.90061	???? PGRMC1	????Xq22-q24
										?169	-3.79	?0.000367	????0.554	????1.088	?165457	Solute carrier family 27 (fatty acid transport protein), the member 2	?? Hs.11729	???? SLC27A2	????15q21.2
?170	-3.77	?0.000389	????0.687	????1.101	?164532	Catalase	?? Hs.395771	???? CAT	????11p13
										?171	-3.77	?0.000401	????0.969	????1.28	?162934	Leucine carboxyl methyltransgerase	?? Hs.8054	???? LCMT	????16p12.3- ????16p12.1
?172	-3.77	?0.000391	????0.583	????0.822	?160051	Lymphocyte plasmotype albumen 1 (L-net matter)	?? Hs.381099	???? LCP1	????13q14.3
										?173	-3.77	?0.000394	????0.701	????0.97	?168394	Glycoloyl-coa dehydrogenase/3-ketone acetyl-coenzyme A thiolase/enoyl-CoA hydratase (three functional proteins), the β subunit	?? Hs.146812	???? HADHB	????2p23
?174	-3.75	?0.000411	????0.964	????1.164	?162323	EST	?? Hs.426542		????4
										?175	-3.75	?0.000419	????0.689	????1.067	?160471	Translation inhibition albumen p14.5	?? Hs.18426	???? UK114	????8q22
?176	-3.75	?0.00042	????0.624	????0.823	?163224	DC2 albumen	?? Hs.103180	???? DC2	????4q25
										?177	-3.73	?0.000444	????0.998	????1.308	?162773	Calcium channel, voltage dependent form, beta 2 subunit base	?? Hs.30941	???? CACNB2	????10p12
?178	-3.73	?0.000454	????0.88	????1.1	?166579	Interleukin 18 acceptor 1	?? Hs.159301	???? IL18R1	????2q12
										?179	-3.72	?0.00046	????0.665	????1.113	?161872	Serine (or halfcystine) protease inhibitor, clade A (α-1 protease inhibitor, antitrypsin), the member 7	?? Hs.76838	???? SERPINA7	????Xq22.2
?180	-3.71	?0.000467	????0.659	????1.159	?162012	Lipoprotein, Lp (a)	?? Hs.119520	???? LPA	????6q26-q27
										?181	-3.71	?0.000469	????0.859	????1.179	?163509	Hermansky-Pudlak syndromes 3	?? Hs.282804	???? HPS3	????3q24
?182	-3.68	?0.000532	????0.523	????0.732	?165011	Tyrosyl albumen sulfotransferase 1	?? Hs.421194	???? TPST1	????7q11.21
										?183	-3.65	?0.000577	????0.649	????0.875	?164314	KIAA1450 albumen	?? Hs.83243	???? KIAA1450	????4q32.1
?184	-3.64	?0.000582	????0.935	????1.054	?162882	RAB3A interaction protein (rabin3) sample albumen 1	?? Hs.13759	???? RAB3IL1	????11q12-q13.1
										?185	-3.62	?0.000636	????0.769	????1.162	?165530	Cytochrome P450, subfamily IIJ (arachidonic acid cyclooxygenase (epoxygenase)) polypeptide 2	?? Hs.152096	???? CYP2J2	????1p31.3-p31.2

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
										?186	-3.59	?0.000679	????0.487	????0.924	?166057	The POU structural domain, 1 class, transcription factor 1 (Pit1, growth hormone factor 1)	?? Hs.89394	?? POU1F1	????3p11
?187	-3.59	?0.000703	????0.95	????1.228	?167868	General transcription factor IIB	?? Hs.258561	?? GTF2B	????1p22-p21
										?188	-3.58	?0.000706	????0.942	????1.096	?167779	General transcription factor IIE, polypeptide 2, β 34kDa	?? Hs.77100	?? GTF2E2	????8p21-p12
?189	-3.58	?0.000727	????0.947	????1.225	?165329	Rab9 effector p40	?? Hs.19012	?? RAB9P40	????9q34.11
										?190	-3.57	?0.000735	????0.62	????1.11	?166857	Profibrinolysin	?? Hs.75576	?? PLG	????6q26
?191	-3.55	?0.000775	????0.838	????1.215	?165788	Potassium inward rectification passage, subfamily J, the member 8	?? Hs.102308	?? KCNJ8	????12p11.23
										?192	-3.55	?0.000778	????0.862	????1.001	?167386	NNMT	??604856 ??(IncytePD)
?193	-3.55	?0.000795	????0.671	????0.802	?163088	The albumen FLJ21918 that supposes	?? Hs.282093	?? FLJ21918	????16q22.1
										?194	-3.55	?0.00079	????0.795	????1.166	?167385	Electron transfer flavoprotein, α polypeptide (glutaric aciduria II)	?? Hs.169919	?? ETFA	????15q23-q25
?195	-3.54	?0.000799	????1.068	????1.459	?169569	Spermidine/spermine N1-Transacetylase	?? Hs.28491	?? SAT	????Xp22.1
										?196	-3.54	?0.000812	????1.04	????1.362	?160982	Ras response element binding protein 1	?? Hs.171942	?? RREB1	????6p25
?197	-3.53	?0.00083	????0.756	????0.967	?166818	Tropomodulin	?? Hs.374849	?? TMOD	????9q22.3
										?198	-3.52	?0.000844	????0.79	????1.057	?164368	Be similar to RIKEN cDNA 1810013D05 gene [homo sapiens], the mRNA sequence	?? Hs.32699		????12
?199	-3.52	?0.000848	????0.609	????1.001	?160667	SODH	?? Hs.878	?? SORD	????15q15.3
										?200	-3.52	?0.000851	????0.713	????0.894	?160956	Albumin A-the 211C6.1 that supposes	?? Hs.28607	?? LOC57149	????16p11.2
?201	-3.52	?0.000858	????0.625	????0.933	?166778	Phosphoenolpyruvate carboxykinase 2 (mitochondrial)	?? Hs.75812	?? PCK2	????14q11.2
										?202	-3.52	?0.000859	????0.958	????1.507	?167552	The membranin 2 that lysosome is relevant	?? Hs.8262	?? LAMP2	????Xq24
?203	-3.51	?0.000891	????1.012	????1.281	?160125	Oncoprotein is translated controlled type 1	?? Hs.401448	?? TPT1	????13q12-q14
										?204	-3.5	?0.000901	????0.991	????1.197	?161606	The Fc fragment of IgG, acceptor, translocator, α	?? Hs.111903	?? FCGRT	????19q13.3
?205	-3.5	?0.000914	????1.005	????1.238	?165593	Stride film 7 superfamily members 1 (in kidney, raising)	?? Hs.15791	?? TM7SF1	????1q42-q43
										?206	-3.5	?0.000915	????1.009	????1.267	?160129	The kinases 2 that MAP/ microtubule avidity is regulated	?? Hs.157199	?? MARK2	????11q12-q13
?207	-3.47	?0.000997	????0.457	????0.74	?168320	Lactate dehydrogenase A	?? Hs.2795	?? LDHA	????11p15.4
										?208	?3.47	?0.000996	????1.098	????0.826	?160605	P311 albumen	?? Hs.142827	?? P311	????5q22.1
?209	?3.48	?0.000971	????0.938	????0.81	?165174	Homo sapiens cDNA FLJ35787 fis, clone TESTI2005672, be similar to very much ubiquinol-cytochrome c reductase mixture core protein 2 before	?? Hs.265591		????16

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
																Body (EC 1.10.2.2), the mRNA sequence
??210	??3.49	?0.000953	????1.083	????0.92	?166833	Solute carrier family 17 (negatively charged ion/HUCEP-8), the member 5	?? Hs.117865	?? SLC17A5	????6q14-q15
										??211	??3.53	?0.000822	????1.111	????0.953	?165413	WIRE albumen	?? Hs.13996	?? WIRE	????17q21.1
??212	??3.56	?0.000759	????1.08	????0.941	?166348	EGF-R ELISA approach substrate 8 associated protein 1s	?? Hs.28907	?? EPS8R1	????19q13.42
										??213	??3.57	?0.000725	????1.048	????0.9	?163115	EST, the medium albumen FLJ20234[homo sapiens who is similar to supposition] [H.sapiens]	?? Hs.119629		????14
??214	??3.59	?0.000684	????1.124	????0.963	?163579	EST	?? Hs.194441		????6
										??215	??3.59	?0.00068	????1.835	????1.306	?161090	KIAA1641 albumen	?? Hs.44566	?? KIAA1641	????2q11.1
??216	??3.6	?0.000688	????1.173	????0.981	?161354	P21/Cdc42/Rac1 activated kinases 1 (STE20 homologue, yeast)	?? Hs.64056	?? PAK1	????11q13-q14
										??217	??3.61	?0.000661	????0.947	????0.82	?162677	People BRCA2 district, mRNA sequence C G011	?? Hs.142907		????13
??218	??3.64	?0.000582	????0.853	????0.653	?161085	Polysaccharase (the DNA orientation), δ 1, catalytic subunit 125kDa	?? Hs.99890	?? POLD1	????19q13.3
										??219	??3.65	?0.000572	????1.141	????0.985	?161518	The H2A histone family, member A	?? Hs.121017	?? H2AFA	????6p22.2-p21.1
??220	??3.65	?0.000571	????1.232	????1.062	?163109	Mitochondrial ribosomal protein L 43	?? Hs.151945	?? MRPL43	????10q24.1-q24.3
										??221	??3.67	?0.000537	????1.014	????0.841	?164845	Sulfur-bearing oxygen is protein structure domain 4 (endoplasmic reticulum) also	?? Hs.154023	?? TXNDC4	????9q22.33
??222	??3.67	?0.000538	????0.677	????0.528	?162564	A kinases (PRKA) ankyrin (yotiao) 9	?? Hs.58103	?? AKAP9	????7q21-q22
										??223	??3.68	?0.000522	????1.301	????1.059	?164727	EST	?? Hs.125038		????8
??224	??3.68	?0.00052	????0.898	????0.781	?161620	The H4 histone family, member A[homo sapiens], the mRNA sequence	?? Hs.278483		????3
										??225	??3.7	?0.000484	????0.976	????0.854	?161334	The protein 20 D7-FC4 that supposes	?? Hs.128702	?? 20D7-FC4	????19q13.3
??226	??3.74	?0.000428	????1.081	????0.871	?163536	The transducer of ERBB2,2	?? Hs.4994	?? TOB2	????22q13.2-q13.31
										??227	??3.77	?0.000411	????1.376	????1.018	?162152	claudin?4	?? Hs.5372	?? CLDN4	????7q11.23
??228	??3.83	?0.00033	????1.138	????0.936	?169742	EST, the medium albumen FLJ20378[homo sapiens who is similar to supposition] [H.sapiens]	?? Hs.143992		????2
										??229	??3.84	?0.000311	????1.234	????1.035	?161058	Multiple endocrine neoplasia I	?? Hs.423348	?? MEN1	????11q13
??230	??3.84	?0.000311	????0.765	????0.619	?161813	KIAA0874 albumen	?? Hs.27973	?? KIAA0874	????18p11.21
										??231	??3.84	?0.000311	????1.227	????1.01	?168511	MutS homologue 2, colorectal carcinoma, non-polyposis type 1 (E.coli)	?? Hs.78934	?? MSH2	????2p22-p21
??232	??3.84	?0.000309	????1.265	????1.031	?161873	Incyte?EST	??3031912

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
																	??(IncytePD)
?233	?3.89	?0.000263	????1.174	????1.002	?169310	The plain 62kDa of nucleopore	?? Hs.9877	?? NUP62	??19q13.33
										?234	?3.9	?0.000259	????2.07	????1.521	?168933	The middle factor (midkine) (axon growth promotes the factor 2)	?? Hs.82045	?? MDK	??11p11.2
?235	?3.96	?0.000213	????1.058	????0.893	?163495	The albumen FLJ11280 that supposes	?? Hs.3346	?? FLJ11280	??1q21.2
										?236	?3.96	?0.000209	????0.749	????0.557	?168500	Homo sapiens cDNA:FLJ21930 fis, clone HEP04301 is similar to HSU90916 people and clones 23815 mRNA sequences very much	?? Hs.82845		??11
?237	?3.96	?0.000207	????1.258	????0.984	?168246	Thyroid Hormone Receptors interaction protein 13	?? Hs.6566	?? TRIP13	??5p15.33
										?238	?3.98	?0.000199	????1.087	????0.914	?164713	Homo sapiens's total length is inserted cDNA clone ZC18H06, mRNA sequence	?? Hs.384561		??19
?239	?4.03	?0.000168	????1.324	????0.862	?169559	E74-like factor 3 (ets structural domain transcription factor, epithelium Idiotype)	?? Hs.166096	?? ELF3	??1q32.2
										?240	?4.04	?0.000162	????1.138	????0.906	?164262	Membranin, palmitoylation type 6 (MAGUK p55 subfamily member 6)	?? Hs.108931	?? MPP6	??7p15
?241	?4.04	?0.000164	????1.187	????0.998	?161661	The albumen FLJ10520 that supposes	?? Hs.77510	?? FLJ10520	??16q22.3
										?242	?4.05	?0.000159	????1.15	????0.926	?163071	Homo sapiens cDNA:FLJ21409 fis, clone COL03924, mRNA sequence	?? Hs.172129		??5
?243	?4.12	?0.000126	????1.058	????0.922	?165465	The KIAA0195 gene product	?? Hs.301132	?? KIAA0195	??17q25.2
										?244	?4.14	?0.000117	????1.265	????1.023	?164085	EST	?? Hs.107845		??2
?245	?4.14	?0.000119	????1.301	????1.049	?166229	The albumen FLJ11362 that supposes	?? Hs.8929	?? FLJ11362	??Xq25-q26.1
										?246	?4.18	?0.000102	????1.027	????0.872	?166228	Huntington protein (huntingtin) (HuntingtonShi disease)	?? Hs.79391	?? HD	??4p16.3
?247	?4.21	?9.10E-05	????0.614	????0.427	?169583	Neural corpuscular protein (neurogranin) (the protein kinase C substrate, RC3)	?? Hs.26944	?? NRGN	??11q24
										?248	?4.3	?6.80E-05	????1.37	????1.01	?160913	claudin?4	?? Hs.5372	?? CLDN4	??7q11.23
?249	?4.31	?6.60E-05	????1.063	????0.844	?168965	Formin conjugated protein 3	?? Hs.107213	?? FNBP3	??2q23.3
										?250	?4.35	?5.80E-05	????1.154	????0.878	?166849	P53-responsive genes 5	??1510581 ??(IncytePD)
?251	?4.37	?5.30E-05	????1.021	????0.816	?167919	KIAA1361 albumen	?? Hs.15119	?? KIAA1361	??17q11.1
										?252	?4.45	?4.00E-05	????1.219	????0.977	?166837	EST	?? Hs.279482		??2
?253	?4.45	?4.10E-05	????1.278	????0.974	?168977	Homo sapiens cDNA FLJ34031 fis, clone FCBBF2003895, mRNA sequence	?? Hs.340316		??19
										?254	?4.49	?3.50E-05	????1.06	????0.93	?166408	The albumen FLJ39514 that supposes	?? Hs.48565	?? FLJ39514	??4q11

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
										?255	?4.49	?3.50E-05	????1.233	????0.952	?167009	Protein kinase C, iota	?? Hs.1904	?? PRKCI	??3q26.3
?256	?4.6	?2.40E-05	????1.237	????1.011	?168029	The ribonucleoprotein polypeptide A of small nut	?? Hs.173255	?? SNRPA	??19q13.1
										?257	?4.61	?2.40E-05	????0.838	????0.632	?169587	V-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homologues	?? Hs.433714	?? KRAS2	??12p12.1
?258	?4.61	?2.40E-05	????1.425	????0.974	?163235	FLJ00005 albumen	?? Hs.367690	?? FLJ00005	??15q22.33
										?259	?4.63	?2.20E-05	????1.153	????0.953	?161066	The albumen of supposing is from clone 24796	?? Hs.27191	?? LOC57146	??16p12
?260	?4.67	?1.90E-05	????0.967	????0.784	?165515	3-phosphoinositide dependent protein kinase-1	?? Hs.154729	?? PDPK1	??16p13.3
										?261	?4.67	?1.90E-05	????1.035	????0.775	?169403	Protein phosphatase 1 is regulated (inhibition) subunit 12 A	?? Hs.16533	?? PPP1R12A	??12q15-q21
?262	?4.71	?1.60E-05	????1.14	????0.951	?169490	The protein D KFZp564K0322 that supposes	?? Hs.97876	?? DKFZP564K0?? 32	??19q13.32
										?263	?4.71	?1.60E-05	????3.6	????1.727	?160089	The calcium signal transducer 1 that tumour is relevant	?? Hs.692	?? TACSTD1	??2p21
?264	?4.73	?1.50E-05	????1.055	????0.889	?169508	The ATP enzyme, transhipment Cu++, α polypeptide (Menkes syndrome)	?? Hs.606	?? ATP7A	??Xq13.2-q13.3
										?265	?4.8	?1.20E-05	????1.478	????1.137	?163214	The albumen FLJ22548 that supposes is similar to gene trap PAT 12	?? Hs.103267	?? FLJ22548	??12q14.3
?266	?5.16	?3.00E-06	????1.12	????0.89	?168509	EST is similar to KHLX_ people Kelch-sample albumin X [H.sapiens] slightly	?? Hs.99398		??14
										?267	?5.17	?3.00E-06	????0.855	????0.668	?166434	The albumen FLJ13213 that supposes	?? Hs.331328	?? FLJ13213	??15q21.2
?268	?5.37	?1.00E-06	????1.164	????0.929	?161233	Incyte?EST	??1602194 ??(IncytePD)
										?269	?5.55	?p＜0.000001	????1.449	????0.963	?167498	Protocalcium MUC-1 7	?? Hs.106511	?? PCDH17	??13q14.3
?270	?5.99	?p＜0.000001	????1.201	????0.896	?160943	Homo sapiens clone 24630 mRNA sequences	?? Hs.171553		??3
										?271	?6.36	?p＜0.000001	????1.345	????1.012	?165379	The protein B C008647 that supposes	?? Hs.102480	?? LOC91875	??14q11.1
?272	?6.36	?p＜0.000001	????1.376	????0.962	?167992	KIAA1557 albumen	?? Hs.6185	?? KIAA1557	??12p11.21
										?273	?6.37	?p＜0.000001	????1.229	????0.824	?166068	Ectoderm neural cortex (having BTB-spline structure territory)	?? Hs.104925	?? ENC1	??5q12-q13.3

Have preceding 25 genes of minimum parameter p value (p＜0.000001), from the set of 273 genes, selected, and the set of these 25 genes can produce and the similar result of the set of 273 genes.This 25 genes (they develop into the risk of HCC aspect significance is arranged), its gene symbol, its chromosome map spectral position, its UG bunch identification number in table 6, have been listed the indication liver problem sufferer.Determined further to suffer from the set that has significant gene to constitute aspect the HCC risk the serious liver problem sufferer of prediction with similar fashion, and in table 7, listed by 10.

Table 6. is used for differentiating 25 remarkable genes of the patient that may suffer from HCC and calculating the required value of multiplefactor L value at predictive model by compound multivariate prediction

	The t-value	Parameter p-value	%CV supports	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
											????1	????-7.28	????0.0000001	????100	????0.603	????0.903	??160198	Cofilin 2 (muscle)	?? Hs.180141	?? CFL2	??14q
????2	????-6.53	????0.0000001	????100	????0.985	????1.607	??168023	The Fc fragment of IgG, high-affinity Ia, acceptor (CD64)	?? Hs.77424	?? FCGR1A	??1q21.2- ??q21.3
											????3	????-6.46	????0.0000001	????100	????0.643	????1.175	??162315	Calcium channel, voltage dependent form, β 3 subunits	?? Hs.250712	?? CACNB3	??12q13
????4	????-6.18	????0.0000001	????100	????0.688	????1.112	??160302	Actomyosin IB	?? Hs.121576	?? MYO1B	??2q12-q34
											????5	????-6.16	????0.0000001	????100	????0.473	????1.161	??169417	Hemocyanin (ferroxidase)	?? Hs.296634	?? CP	??3q23-q25
????6	????-6.1	????0.0000001	????100	????0.876	????1.18	??161756	Albumin	?? Hs.184411	?? ALB	??4q11-q13
											????7	????-6.04	????0.0000001	????100	????0.719	????1.224	??162290	UDP-N-acetyl glucosamine pyrophosphorylase 1	?? Hs.21293	?? UAP1	??1q23.1
????8	????-6.01	????0.0000001	????100	????0.534	????1.141	??162538	Unknown [homo sapiens], the mRNA sequence	?? Hs.367982		??16
											????9	????-5.94	????0.0000001	????100	????0.491	????0.714	??168634	Karyomit(e) 20 open reading frame 3	?? Hs.22391	?? C20orf3	??20p11.22- ??p11.21
????10	????-5.93	????0.0000001	????100	????0.756	????1.276	??164136	Acetyl-coa dehydrogenase, long-chain	?? Hs.1209	?? ACADL	??2q34-q35
											????11	????-5.9	????0.0000001	????100	????0.864	????1.181	??163874	The KIAA0092 gene product	?? Hs.151791	?? KIAA0092	??11q21
????12	????-5.88	????0.0000001	????100	????0.728	????0.925	??163096	CGI-26 albumen	?? Hs.24332	?? CGI-26	??12p12.3
											????13	????-5.73	????0.0000001	????100	????0.616	????1.133	??160233	The kinases 3 that dual specific tyrosine-(Y)-phosphorylation is regulated	?? Hs.38018	?? DYRK3	??1q32
????14	????-5.67	????0.0000001	????100	????0.786	????1.071	??160436	Be similar to the albumen PRO2831[homo sapiens of supposition], the mRNA sequence	?? Hs.406646		??15

	The t-value	Parameter p-value	%CV supports	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
											??15	???-5.65	??0.0000001	??100	????0.761	????1.382	??160795	The liver leukemia factor	?? Hs.433707	?? HLF	??17q22
??16	???-5.61	??0.0000001	??100	????0.314	????0.798	??161944	Complement component 9	?? Hs.1290	?? C9	??5p14-p12
											??17	???-5.6	??0.0000001	??100	????0.506	????0.703	??167718	ATP-binding cassette, subfamily A (ABC1), the member 1	?? Hs.211562	?? ABCA1	??9q31.1
??18	???-5.58	??0.0000001	??100	????0.65	????0.912	??168437	KIAA0843 albumen	?? Hs.26777	?? KIAA0843	??5q32
											??19	???-5.57	??0.0000001	??100	????0.843	????1.087	??162884	The Phospholipase A2 γ that does not rely on calcium that intracellular membrane is relevant	?? Hs.44198	?? IPLA2(GAM?? MA)	??7q31
??20	???-5.48	??0.0000001	??100	????0.657	????1.065	??166910	SIPL albumen	?? Hs.64322	?? SIPL	??2p25.3
											??269	????5.55	??0.0000001	??100	????1.449	????0.963	??167498	Protocalcium MUC-1 7	?? Hs.106511	?? PCDH17	??13q14.3
??270	????5.99	??0.0000001	??100	????1.201	????0.896	??160943	Homo sapiens clone 24630mRNA sequence	?? Hs.171553		??3
											??271	????6.36	??0.0000001	??100	????1.345	????1.012	??165379	The protein B C008647 that supposes	?? Hs.102480	?? LOC91875	??14q11.1
??272	????6.36	??0.0000001	??100	????1.376	????0.962	??167992	KIAA1557 albumen	?? Hs.6185	?? KIAA1557	??12p11.21
											??273	????6.37	??0.0000001	??100	????1.229	????0.824	??166068	Ectoderm neural cortex (having BTB-spline structure territory)	?? Hs.104925	?? ENC1	??5q12-q13.3

These 25 genes are selected by minimum parameter p value (p＜0.000001).

Table 7. is used for predicting 10 remarkable genes of suffering from HCC and calculates the required value of multiplefactor L value at predictive model

	The t-value	Parameter p-value	The geometric mean of ratio in excessive risk group 1	The geometric mean of ratio in low risk group 2	High/low	Unique label	Describe	UG bunch	Gene symbol	Chromosome position
											????103	??-4.26	?7.70E-05	????0.406	????0.89	??0.45618	?161362	Tryptophane 2,3-dioxygenase enzyme	? Hs.183671	?? TDO2	????4q31-q32
????77	??-4.57	?3.60E-05	????0.421	????0.88	??0.478409	?161748	Acetyl-CoA Transacetylase 1 (acetate acetyl-CoA thiolase)	? Hs.37	?? ACAT1	????11q22.3-q23.1
											????42	??-5.02	?5.00E-06	????0.506	????1.137	??0.445031	?162311	Serine (or halfcystine) protease inhibitor, clade C (antithrombin), the member 1	? Hs.75599	?? SERPINC1	????1q23-q25.1
????8	??-6.01	?p＜0.000001	????0.534	????1.141	??0.468011	?162538	Unknown [homo sapiens], the mRNA sequence	? Hs.367982		????16
											????63	??-4.7	?1.80E-05	????0.355	????0.917	??0.387132	?162617	FK506 conjugated protein 5	? Hs.7557	?? FKBP5	????6p21.3-21.2
????131	??-4.05	?0.000155	????0.565	????1.142	??0.494746	?162666	Prokinin	? Hs.77741	?? KNG	????3q27
											????121	??-4.14	?0.000116	????0.59	????1.176	??0.501701	?164863	Solute carrier family 2 (auxiliary glucose transporter), the member 2	? Hs.167584	?? SLC2A2	????3q26.1-q26.2
????142	??-3.97	?0.000205	????0.418	????0.896	??0.466518	?166007	Tyrosine aminotransferase	? Hs.161640	?? TAT	????16q22.1
											????116	??-4.19	?9.90E-05	????0.507	????1.102	??0.460073	?169375	Cytochrome P450, subfamily IIC (mephenytoin 4-hydroxylase), polypeptide 9	? Hs.167529	?? CYP2C9	????10q24
????5	??-6.16	?p＜0.000001	????0.473	????1.161	??0.407407	?169417	Hemocyanin (ferroxidase)	? Hs.296634	?? CP	????3q23-q25

Claims (45)

1. differentiate the method that suppresses the potential treatment target spot of cancer metastasis among the hepatocellular carcinoma HCC patient for one kind, it is characterized in that, may further comprise the steps:

A) will contact with the chip that comprises at the capture agent of one group of cell sign thing from transitivity HCC patient's sample;

B) from sample, catch mark and produce first signal;

C) thus with the HCC patient's of non-transfer sample repeating step a) and step b) produce second signal;

D) compare first and second signals, thereby identify the different cell sign thing subgroup of level of first signal and second signal, this subgroup cell marker is exactly the potential treatment target spot that treatment patient HCC HCC shifts.

2. the method for claim 1 is characterized in that,

The signal that the normal non-cancer tissue sample of deduction is produced on the chip identical with the chip of step a) in step b) and step c), thus first and second signals produced.

3. a method of predicting hepatocellular carcinoma HCC patient cancer metastasis is characterized in that, comprises the steps:

A) will contact with the chip that comprises at the capture agent of one group of cell sign thing from transitivity HCC patient's sample, this group cell sign thing comprises at least 10 genes independently selecting or the albumen of coded by said gene from table 2 gene;

B) from sample, catch mark;

C) from the mark that is hunted down of step b), produce first signal;

D) thus a) produce second signal to step c) with the HCC patient's of non-transfer sample repeating step;

E) thus shift possible HCC patient's sample repeating step and a) produce the 3rd signal to step c) with indeterminate having or not;

F) the 3rd signal and first and second signals are compared, thus determining step e) HCC patient's metastatic potential.

4. method as claimed in claim 3 is characterized in that, this group cell sign thing comprises at least 20 genes independently selecting or the albumen of coded by said gene from table 2 gene.

5. method as claimed in claim 4 is characterized in that, this group cell sign thing comprises at least 50 genes independently selecting or the albumen of coded by said gene from table 2 gene.

6. method as claimed in claim 5 is characterized in that, this group cell sign thing comprises at least 100 genes independently selecting or the albumen of coded by said gene from table 2 gene.

7. method as claimed in claim 6 is characterized in that, this group cell sign thing comprises the gene of table 2 or the albumen of coded by said gene.

8. method as claimed in claim 3 is characterized in that, this group cell sign thing comprises the gene of table 4 or the albumen of coded by said gene.

9. method as claimed in claim 3, it is characterized in that this group cell sign thing comprises that single-gene is numbered the gene of Hs.313, Hs.69707, Hs.222, Hs.63984, Hs.75573, Hs.177687, Hs.69707, Hs.222, Hs.323712 and Hs.63984 or the albumen of coded by said gene.

10. method as claimed in claim 3 is characterized in that, step a) and b) sample, the sample of step d) and the sample of step e) be the hepatic tissue extract.

11. method as claimed in claim 3 is characterized in that, the chip in the step a) is a genome chip.

12. method as claimed in claim 3 is characterized in that, the chip in the step a) is the protein group chip.

13. a discriminating is used to prevent that chronic hepatitis patients from developing into the method for the potential treatment target spot of hepatocellular carcinoma HCC, it is characterized in that, may further comprise the steps:

A) will contact with the chip that comprises at the capture agent of one group of cell sign thing from the sample of the high-risk chronic hepatitis patients of HCC;

B) from sample, catch mark and produce first signal;

C) a) and step b), with the chronic hepatitis patients sample repeating step of the low danger of HCC thus produce second signal;

D) compare first and second signals, thereby identify the different cell sign thing subgroup of level of first signal and second signal, the cell marker of this subgroup is exactly to prevent that chronic hepatitis patients from developing into the potential treatment target spot of HCC.

14. as method as described in the claim 13, it is characterized in that, the signal that the normal non-cancer tissue sample of deduction is produced on the chip identical with the chip of step a) in step b) and step c), thus produce first and second signals.

15. predict that chronic hepatitis patients develops into the method for the danger of hepatocellular carcinoma HCC for one kind, it is characterized in that, may further comprise the steps:

A) will contact with the chip that comprises at the capture agent of one group of cell sign thing from the sample of the high-risk chronic hepatitis patients of HCC, this group cell sign thing comprises the albumen by at least 10 genes independently selecting in table 5 gene or coded by said gene;

B) from sample, catch mark;

C) from the captive mark of step b), produce first signal;

D) thus a) produce second signal to step c) with the chronic hepatitis patients sample repeating step of the low danger of HCC;

E) thus a) produce the 3rd signal to step c) with the chronic hepatitis patients sample repeating step of the indeterminate HCC of having or not danger;

F) the 3rd signal and first, second signal are compared, thus determining step e) the patient develop into the danger of HCC.

16. method as claimed in claim 15 is characterized in that, this group cell sign thing comprises at least 20 genes independently selecting or the albumen of coded by said gene from table 5 gene.

17. method as claimed in claim 16 is characterized in that, this group cell sign thing comprises at least 50 genes independently selecting or the albumen of coded by said gene from table 5 gene.

18. method as claimed in claim 17 is characterized in that, this group cell sign thing comprises at least 100 genes independently selecting or the albumen of coded by said gene from table 5 gene.

19. method as claimed in claim 18 is characterized in that, this group cell sign thing comprises the gene of table 5 or the albumen of coded by said gene.

20. method as claimed in claim 15 is characterized in that, this group cell sign thing comprises the gene of table 6 or the albumen of coded by said gene.

21. method as claimed in claim 15 is characterized in that, this group cell sign thing comprises the gene of table 7 or the albumen of coded by said gene.

22. method as claimed in claim 15 is characterized in that, step a) and b) sample, the sample of step d) and the sample of step e) be the hepatic tissue extract.

23. method as claimed in claim 15 is characterized in that, the chip in the step a) is a genome chip.

24. method as claimed in claim 15 is characterized in that, the chip in the step a) is the protein group chip.

25. method as claimed in claim 15 is characterized in that, the disease that the patient suffered from the step a) is selected from down group: hepatitis B, third liver, hemochromatosis and WilsonShi disease.

26. method as claimed in claim 15 is characterized in that, the disease that the patient suffered from the step d) is selected from down group: alcoholic liver disease, autoimmune hepatitis and primary biliary cirrhosis.

27. method as claimed in claim 15 is characterized in that, the disease that patient suffered from the step e) is selected from down group: hepatitis B, third liver, hemochromatosis, WilsonShi disease, alcoholic liver disease, autoimmune hepatitis and primary biliary cirrhosis.

28. a computer-readable medium is characterized in that, comprising:

A) code of first data set, this data set derives from first signal, the chip that this signal contacts from the sample with transitivity HCC patient, described chip comprises the capture agent at one group of cell sign thing, and this group cell sign thing comprises the albumen by at least 10 genes independently selecting in table 2 gene or coded by said gene;

B) code of second data set, this data set derives from second signal, the chip that this signal contacts from the sample with non-metastatic HCC patient, described chip is identical with a) chip;

C) code of the 3rd data set, this data set derives from the 3rd signal, the chip that this signal contacts from the HCC patient's who shifts with the unknown sample, described chip is identical with a) chip;

D) code that the 3rd data set and first and second data sets are compared.

29. a digital computer is characterized in that, it comprises the described computer-readable medium of claim 28.

30. a system is characterized in that, comprising:

A) the described digital computer of claim 29;

B) comprise chip at the array of the capture agent of one group of cell sign thing, described mark comprises at least 10 genes independently selecting or the albumen of coded by said gene from table 2 gene;

C) the back reader that read signal from chip can contacted with sample.

31. a computer-readable medium is characterized in that it comprises:

A) code of first data set, this data set derives from first signal, the chip that this signal contacts from the patient's high-risk with suffering from chronic hepatopathy and HCC sample, described chip comprises the capture agent at one group of cell sign thing, and this group cell sign thing comprises the albumen by at least 10 genes independently selecting in table 5 gene or coded by said gene;

B) code of second data set, this data set derives from second signal, and this signal is from the chip that contacts with the patient's of the low danger of chronic hepatopathy and HCC sample, and described chip is identical with a) chip;

C) code of the 3rd data set, described data set derives from the 3rd signal, and this signal is from chronic hepatopathy and develop into the chip that patient's the sample of risk level the unknown of HCC contacts, and described chip is identical with a) chip;

D) code that the 3rd data set and first and second data sets are compared.

32. a digital computer is characterized in that, it comprises the described computer-readable medium of claim 31.

33. a system is characterized in that, comprising:

A) the described digital computer of claim 32;

B) comprise chip at the array of the capture agent of one group of cell sign thing, described mark comprises at least 10 genes independently selecting or the albumen of coded by said gene from table 5 gene;

C) the back reader that read signal from chip can contacted with sample.

34. a method that suppresses metastasis of cancer among the hepatocellular carcinoma HCC patient is characterized in that the method comprising the steps of: suppress osteopontin (OPN) activity.

35. method as claimed in claim 34 is characterized in that, suppressing the active step of osteopontin is to express by inhibition OPN to finish.

36. method as claimed in claim 35 is characterized in that, is used for suppressing the expression of OPN with antisense polynucleotides.

37. method as claimed in claim 34 is characterized in that, suppressing the active step of osteopontin is to finish by the specific combination that suppresses between OPN and the OPN acceptor.

38. method as claimed in claim 37 is characterized in that, employing OPN antagonist suppresses the specific combination between OPN and the OPN acceptor.

39. method as claimed in claim 37 is characterized in that, adopts anti-OPN antibody to suppress specific combination between OPN and the OPN acceptor.

40 1 kinds are suppressed the method that chronic hepatitis patients develops into hepatocellular carcinoma HCC, it is characterized in that, comprise step: the activity that suppresses EpCAM.

41. method as claimed in claim 40 is characterized in that, suppressing the active step of EpCAM is to express by inhibition EpCAM to finish.

42. method as claimed in claim 41 is characterized in that, adopts antisense polynucleotides to suppress the expression of EpCAM.

43. method as claimed in claim 41 is characterized in that, adopts little inhibitory RNA to suppress the expression of EpCAM.

44. method as claimed in claim 40 is characterized in that, suppressing the active step of EpCAM is to finish by the specific combination that suppresses between EpCAM and the EpCAM acceptor.

45. method as claimed in claim 44 is characterized in that, employing anti-EpCAM antibody suppresses the specific combination between EpCAM and the EpCAM acceptor.

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