CN101104871A - Mammary cancer marker gene group and application method thereof - Google Patents

Abstract

This invention discloses a marked gene group of breast cancer and an application approach on diagnosis of breast benign and malignant tumors by combination of one, a plurality of genes or all genes in the group. The gene group has 120 genes which relates to key biological process pertinent to breast cancer occurrence such as growth and metabolism of cell, cell signaling, cell adhesion motion and immunity and so on. The application approach can be gene chip, molecular hybridization and RT-PCR and so on to detect the mRNA expression of the genes in a sample tissue, which can identify the sample tissue of a patient to be benign and malignant according to the gene expression.

Description

Mammary cancer marker gene group and application method thereof

Technical field

The present invention relates to the express spectra research and the marker gene group screening of the disease related gene of functional genomics.Specifically be the marker gene group of screening mammary cancer in people's functional gene class range, and use gene chip, methods such as molecular hybridization and RT-PCR to detect the mRNA expression level of these genes, differentiate the good, pernicious of breast tumor according to the mRNA expression level.

Technical background

Mammary cancer is the modal malignant tumour of women, is the significant threat of WomanHealth, and early diagnosis is the key that improves mammary cancer curative ratio and survival rate.Tumour is the unusual disease of polygene, and tumor tissue cell has heterogeneous biological property, in the generating process of mammary cancer, relate to the disorder of the inactivation, cellular metabolism and the cell cycle regulating that comprise oncogene active and cancer suppressor gene, the biological procedures that apoptotic inhibition etc. are complicated.Found that at present over one hundred kind of tumour promotes gene such as K-ras, c-myc, c-erbB-2 etc. are relevant with oncogenesis with tens kinds of tumor suppressor genes such as P53, Rb, P16 etc., but individual gene detects or the joint-detection of part known still can not be carried out diagnosis and differential diagnosis to breast cancer tissue's sample and healthy tissues sample.Biochip technology is can the parallel detection of high-throughput thousands of to tens thousand of expression of gene levels, thereby can in genome range, study histiocytic gene expression profile, screening obtains the marker gene group that malignant tumour is different from healthy tissues, is used for the diagnosis and differential diagnosis of tumour.

Summary of the invention

The present invention adopts mammary gland primary carcinoma and the paired cancer other normal galactophore tissue gene expression difference thereof of high-throughout people's expression profiles of gene chip by many cases, screening obtains the marker gene group of one group of mammary cancer, detect in the marker gene group one, the mRNA level of a plurality of or full gene with methods such as gene chip, molecular hybridization, RT-PCR, and then breast tumor is made differential diagnosis.Main invention is as follows:

1. one group of mammary cancer marker gene group is characterized by in this group gene group one, combination a plurality of or full gene and can be used to differentiate that mammary gland is good, malignant tumour, and the title of 120 genes, sequence number and major function are described and seen Table 1 in the gene group.

2. the combined preparation of a plurality of or full gene of the described gene of claim 1 becomes that gene chip is used for very, the evaluation of malignant breast tumor, it is characterized in that the cDNA or the oligonucleotide probe of these genes are fixed into microarray, wherein each gene probe can be hybridized with the cDNA that derives from corresponding gene in the tissue samples or its amplified production specifically, by classification, microarray can be flat board, microballon, fine needle or film phase array, can be solid phase, liquid phase; Can be oligonucleotide, polynucleotide or cDNA array.

3. the single or multiple genes of the described gene of claim 1 detect mRNA expression amount in the tissue sample as the goal gene of the probe of making nucleic acid molecular hybridization or RT-PCR amplification, identify good, malignant tumour according to the mRNA expression level, making nucleic acid molecular hybridization can be hybridization on solid support of the cDNA of the RNA that extracts and reverse transcription thereof, also can be the in situ hybridization of tissue slice that RT-PCR can be quantivative approach, semi-quantitative method, qualitative method.

4. utilize the described gene chip of claim 2 to differentiate mammary cancer and healthy tissues or carcinoid method:

(1) behind the cDNA of breast tumor tissues or healthy tissues sample or its amplified production fluorescent mark with comprise the gene chip hybridization of a plurality of or full gene of mammary cancer marker gene group, based on the fluorescence intensity level of each gene, utilizing non-supervision clustering method promptly to obtain can be with the gene expression profile of breast cancer tissue and the accurate classification of healthy tissues;

(2) use gene expression atlas that breast tumor is carried out differential diagnosis, may further comprise the steps: may further comprise the steps:

A) from breast tumor patient's excision sample or needle biopsy sample, extract total RNA and reverse transcription is cDNA;

B) cDNA or its amplified production to sample carries out fluorescent mark;

C) with sample behind the mark and the gene chip hybridization described in the claim 2

D), relatively patient tissue sample good, pernicious made evaluation with the gene mapping described in the claim 4 with non-supervision cluster analysis sample gene expression profile.

Description of drawings

Fig. 1 is based on the cluster of differential gene group to 9 routine mammary gland primary carcinoma and pairing normal galactophore tissue;

Fig. 2 is based on the cluster result of differential gene group to checking sample and learning sample.

Embodiment

1 technical measures

1.1 case is selected and sample disposal

The case of the capable radical mastectomy that 24 routine patient with breast cancers accept for medical treatment for Tumour Hospital Attached To Tianjin Medical Univ., histological type is infitrating ductal carcinoma (WHO classification), and the tumour size is 2～5 centimetres (T2 phases); Histological grade I level 1 example, II level 19 examples, III level 4 examples; Positive 15 examples of nodus lymphoideus transferring rate, negative 10 examples; Positive 17 examples of ER, negative 6 examples; Positive 11 examples of PR, negative 12 examples.The cancerous tissue sample confirms through histopathologic diagnosis, and the other normal galactophore tissue of cancer gets apart from the normal gland tissue more than the borderline tumor 5cm, specimen sampling after liquid nitrogen flash freezer in-80 ℃ of preservations.In addition, get 32 routine breast tumor patients' normal galactophore tissue, extract behind total RNA balanced mix as the check sample of gene chip hybridization.

1.2 gene chip hybridization

1.2.1 total RNA extracts: adopt Trizol (Invitrogen company, the U.S.) single stage method is extracted histiocytic total RNA, RNeasy mini spin column Kit (Qiagen company, the U.S.) purifying RNA, the purity of determined by ultraviolet spectrophotometry RNA, agarose gel electrophoresis detects the integrity of RNA.

1.2.2 double-stranded cDNA is synthetic: get the total RNA of 10 μ g, with T7-Oligo (dT) 15, (5 '-AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCTTTTTTTTTTTTTT TTV-3, V is G, C and A, Shanghai Bo Ya Bioisystech Co., Ltd) be primer, with cDNASynthesis Kit (TaKaRa, China) synthetic double chain cDNA, the double-stranded cDNA of QIAquick PCRPurification Kit (Qiagen company, the U.S.) purifying.

1.2.3 in-vitro transcription is synthesized cRNA: with T7 RiboMAX Express Large Scale RNAProduction System (Promega company, the U.S.) double-stranded cDNA is carried out the synthetic cRNA of in-vitro transcription, and use RNeasy Mini Kit (Qiagen company, the U.S.) purifying.

.1.2.4 the reverse transcription of cRNA: get 2 μ g cRNA, carry out reverse transcription, QIAquick PCRPurification Kit purifying reverse transcription product with random primer and SuperscriptII ThermoScript II (Invitrogen company, the U.S.).

1.2.5 the fluorescent mark of cDNA: get 1 μ g cRNA reverse transcription product, Random PrimerDNA Labeling Kit (TaKaRa, China) mark fluorescent, Cy3 mark normal control sample, Cy5 labelling experiment group sample (Cy5-dCTP, Cy3-dCTP, Amersham PharmaciaBiotech, the U.S.).QIAquick PCR Purification Kit purifying marked product.

1.2.6 gene chip hybridization: people's expression profiles of gene chip is the people Oligo chip of Beijing biochip rich difficult to understand company limited preparation.All genes are taken from the Human genome 70mer Oligo storehouse of Qiagen company on the chip, point sample is at a 75 * 25mm, through on the slide glass of chemically modified, contain about 23232 gene points, 21329 individual function genes, 12 house-keeping genes that also comprise the people are as positive control, the 70 mer Oligo DNA that 12 artificial synthetic and people's gene do not have homology are as negative control, and 3 genes of Arabidopis thaliana are as external standard.Whole dot matrix is divided into 48 inferior battle arrays, and each inferior battle array has 22 row, 22 row.Dot spacing is 185 μ m, and the diameter of point is about 140 μ m.Gene chip is through 65 ℃ of hydrations, and 250mJ is UV-crosslinked, and 0.5%SDS and dehydrated alcohol clean, and is used for hybridization behind the centrifuge dripping.Cy3 and the fluorescently-labeled product mixing of Cy5 are dissolved in the 30 μ L hybridization solutions (3 * SSC, 0.2%SDS, 5 * Denhart ' s, 25% methane amide), and 42 mouthfuls of hybridization are spent the night.Gene chip after the hybridization is developed a film in containing 2 * SSC of 0.2%SDS, 0.2 * SSC and pure water successively, carry out fluorescence intensity scanning behind the centrifuge dripping.

1.2.7 chip scanning and data analysis: fluorescently-labeled gene chip scans with ScanArrayExpress two channels laser scanner (Packard Bioscience company, the U.S.).Adopt GenePix Pro 4.0 image analysis software (Axon company, the U.S.) that chip image is analyzed, fluorescence intensity adopts the Lowess method to carry out stdn.The fluorescence intensity level of one of Cy3 or Cy5 greater than 100 be the effective expression gene.

1.3 differential gene screening: screen in 9 routine analyzing samples primary carcinoma and its paired normal galactophore tissue that at least 6 pairs of samples the gene of same difference expression trend more than 3.0 times to be arranged be difference expression gene.Function [2] with GoMiner software retrieval difference expression gene.

1.4 hierarchial-cluster analysis (Hierachical clustering): adopt cluster software simultaneously 18 learning sample of 9 routine mammary cancer primary carcinoma and the other normal galactophore tissue of paired cancer to be averaged cluster analysis based on the differential gene group.Use other 15 routine mammary gland primary carcinoma as verifying that sample and 18 common clusters of analyzing samples are with the reliability of checking differential gene group to the cancerous tissue sample classification.

2 results judge

2.1 difference expression gene

The other normal galactophore tissue of 9 routine mammary gland primary carcinoma and its paired cancer gene expression profile comparison 6 examples are above greater than 120 of 3.0 times of genes that common differential expression trend arranged, wherein 35 rises, 85 downward modulations; 34 genes are relevant with the cell growth metabolism, 22 relevant with signal conduction and transcriptional regulatory, and 12 relevant with the cell adhesion motion, and 5 relevant with cytodifferentiation, 5 relevant with body immunity, also has the gene of 8 other functions and the gene of 34 unknown function.See Table 1.

2.2 difference expression gene is to the sample cluster analysis result

2.2.1 can exactly 17 samples in 18 analyzing samples of 9 routine mammary cancer accurately be divided into normal and two groups of mammary cancer based on 120 difference expression gene groups, have only 1 normal sample to be in the cancer group.The results are shown in Figure 1.

.2.2.3 based on 120 difference expression gene groups checking sample and 18 analyzing samples of 15 routine mammary gland primary carcinoma are carried out hierarchial-cluster analysis simultaneously, 15 example checking cases accurately are sorted in the mammary cancer group as a result, as Fig. 2.The checking result confirms can accurately judge sample based on the gene expression profile of this gene group of organizing to be checked.

Technical superiority

The generation of tumour cell and development have related to complicated biological procedures, the abnormal change that functional gene is expressed is the basis of cell phenotype vicious transformation, the present invention is by comparing the other normal galactophore tissue of breast cancer patients and cancer gene expression difference, screening has the differential gene of co expression trend in multiple-case, they have related to the important biomolecule of tumour cells such as cell growth metabolism, signal conduction and transcriptional regulatory, cell adhesion motion, cytodifferentiation and immunity of organism generation development and have learned process.Constitute the marker gene group of one group of mammary cancer, mammary cancer and healthy tissues accurately can be sorted in different two groups.The gene group of this invention is one group of cancer associated gene that is woven with common differential expression trend in the breast cancer tissue and the other normal group of cancer of multiple-case, this group gene group energy embodies the unusual biology general character of human breast cancer tissue gene, and the detection method of the mRNA expression level of setting up based on single, a plurality of or full gene wherein can be used for the molecular diagnosis and the differential diagnosis good, malignant tumour of mammary cancer.

The list of genes of table 1 mammary cancer marker gene group (totally 120 genes)

The gene title	The GenBank sequence number	Gene is described
			RGS2 SULF1 TFEC TP73L DMN CKS2 ASPM TMLHE KLK7 SPG3A BBOX1 KLK5 MYLK GZMA GZMB CCNB2 STAT1 MIA DUSP6 H2AFX RERG RRM2 IGFBP1 NEDD9 KIT MAB21L1 HAS3 NCF2 LPL AK5 ANXA3 ABCA6	NM_002923 AB029000 NM_012252 Y16961 AB002351 NM-001827 AK001379 NM_018196 NM_005046 AF444143 NM_003986 NM_012427 NM_053025 NM_006144 NM_004131 NM_004701 NM_007315 NM_006533 NM_001946 NM_002105 NM_032918 NM_001034 NM_000596 NM_006403 NM_000222 NM_005584 AF232772 NM_000433 NM_000237 NM_012093 NM_005139 AY028898	Regulator of G-protein signalling 2，24kD KIAA1077 protein Transcription factor EC Tumor protein 63 kDa with strong homology to p53 Desmuslin CDC28 protein kinase 2 Hypothetical protein FLJ10517 Epsilon-trimethyllysine hydroxylase Kallikrein 7(chymotryptic，stratum comeum) Spastic paraplegia 3A(autosomal dominant) Butyrobetaine (gamma)， 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase)1 Kallikrein 5 Myosin，light polypeptide kinase Granzyme A(granzyme 1，cytotoxic T-lymphocyte-associated serine esterase 3) Granzyme B(granzyme 2，cytotoxic T-lymphocyte-associated serine esterase 1) Cyclin B2 Signal transducer and activator of transcription 1，91kD Melanoma inhibitory activity Dual specificity phosphatase 6 H2A histone family，member X RAS-like，estrogen-regulated，growth-inhibitor Ribonucleotide reductase M2 polypeptide Insulin-like growth factor binding protein 1 Enhancer of filamentation 1(cas-like docking；Crk-associated substrate related) V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog Mab-21-like 1(C.elegans) Hyaluronan synthase 3 Neutrophil cytosolic factor 2(65kD，chronic granulomatous disease， autosomal 2) Lipoprotein lipase Adenylate kinase 5 Annexin A3 Homo sapiens ATP-binding cassette A6 mRNA，complete cds

ABCB1 SCGB2A2 ZAP70 CDCA1 FLG NTRK2 DCX CXCL10 COL1A2 COL11A1 FN1 COL5A1 COL10A1 SRPX MYBPC1 ATP1A2 CX3CL1 CXCL2 MMP3 C11ORF8 LILRB4 SAA4 UBD AOX1 CLDN8 DNER MME PIK3C2G CD3D CXCL9 ANGPTL1 ID4 TCN1 BAIAP1 MEGF10

NM_000927 NM_002411 L05148 NM_031423 M60502 AJ420458 NM_000555 NM_001565 NM_000089 NM_001854 NM_002026 NM_000093 NM_000493 NM_006307 NM_002465 NM_000702 NM_002996 NM_002089 NM_002422 NM_001584 NM_006847 NM_006512 NM_006398 NM_001159 AK022269 AL137311 NM_007289 NM_004570 NM_000732 NM_002416 NM_004673 BC014941 NM_001062 AK023358 NM_032446

ATP-binding cassette，sub-family B(MDR/TAP)，member 1 Mammaglobin 1 Zeta-chain(TCR)associated protein kinase(70kD) Hypothetical protein NUF2R Filaggrin Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 1630957 Doublecortex；lissencephaly，X-linked(doublecortin) Small inducible cytokine subfamily B(Cys-X-Cys)，member10 Collagen，type I，alpha 2 Collagen，type XI，alpha 1 Fibronectin 1 Collagen，type V，alpha 1 Collagen，type X，alpha 1(Schmid metaphyseal chondrodysplasia) Sushi-repeat-containing protein，X chromosome Myosin binding protein C，slow type ATPase，Na+/K+transporting，alpha 2(+)polypeptide Small inducible cytokine subfamily D(Cys-X3-Cys)，member 1 (fractalkine，neurotactin) GRO2 oncogene Matrix metalloproteinase 3(stromelysin 1，progelatinase) Chromosome 11 open reading frame 8 Leukocyte immunoglobulin-like receptor，subfamily B(with TM and ITIM domains)，member 4 Serum amyloid A4，constitutive Diubiquitin Aldehyde oxidase 1 Claudin 8 Homo sapiens mRNA；cDNA DKFZp761G02121(from clone DKFZp761G02121)；partial cds Membrane metallo-endopeptidase(neutral endopeptidase， enkephalinase，CALLA，CD10) Phosphoinositide-3-kinase，class 2，gamma polypeptide CD3D antigen，delta polypeptide(TiT3 complex) Monokine induced by gamma interferon Angiopoietin-like 1 Inhibitor of DNA binding 4，dominant negative helix-loop-helix protein Transcobalamin I(vitamin B12 binding protein，R binder family) Homo sapiens cDNA FLJ13296 fis，clone OVARC1001261 MEGF10 protein

PIGR ARHE LAIR2 OGN LEPR ADAMDEC1 XLKD1 CCL28 HCLS1 LDB2 KLF5 KCNJ16 SYNPO2 KRT17 KRT5 MYH11 FABP4 C2 MATN2 FLJ30296 TU3A CTHRC1 DKFZP586H 2123 FLJ32771 DKFZP586C0 721 MGC21981 LOC338769 C20ORF82 BPAG1 KRT15 B7-H4 FLJ13710 ECRG4 ODZ2 HN1

AK026320 BC012513 NM_002288 NM_033014 U50748 NM_014479 NM_006691 NM_019846 NM_005335 NM_001290 NM_001730 NM_018658 AK021484 NM_000422 NM_000424 NM_022844 NM_001442 NM_000063 NM_002380 AK054858 NM_007177 BC014245 AK027841 AK057333 AL137734 BC015417 BC017984 BC017997 NM_001723 NM_002275 NM_024626 NM_024817 NM_032411 AB032953 NM_016185

Homo sapiens cDNA：FLJ22667 fis，clone HSI08385 Ras homolog gene family，member E Leukocyte-associated Ig-like receptor 2 Osteoglycin(osteoinductive factor，mimecan) Leptin receptor ADAM-like，decysin 1 Extracellular link domain-containing 1 CC chemokine CCL28 Hematopoietic cell-specific Lyn substrate 1 LIM domain binding 2 Kruppel-like factor 5(intestinal) Potassium inwardly-rectifying channel，subfamily J，member 16 Homo sapiens cDNA FLJ11422 fis，clone HEMBA1001008 Keratin 17 Keratin 5(epidermolysis bullosa simplex， Dowling-Meara/Kobner/Weber-Cockayne types) Myosin，heavy polypeptide 11，smooth muscle Fatty acid binding protein 4，adipocyte Complement component 2 Matrilin 2 Homo sapiens cDNA FLJ30296 fis，clone BRACE2003110，weakly similar to PATCHED PROTEIN HOMOLOG 1 TU3A protein Homo sapiens，Similar to RIKEN cDNA 1110014B07 gene，clone MGC：20766 IMAGE：4586039，mRNA，completec DKFZP586H2123 protein Homo sapiens cDNA FLJ3277 1 fis，clone TESTI2001950 Homo sapiens mRNA；cDNA DKFZp586C0721(from clone DKFZp586C0721)；partial cds Homo sapiens，clone MGC：21981 IMAGE：4396073，mRNA， complete cds Homo sapiens，clone IMAGE：4245930，mRNA Homo sapiens，clone IMAGE：4252124，mRNA，partial cds Bullous pemphigoid antigen 1(230/240kD) Keratin 15 Hypothetical protein FLJ22418 Hypothetical protein FLJ13710 Esophageal cancer related gene 4 protein Odd Oz/ten-m homolog 2(Drosophila，mouse) Hematological and neurological expressed 1

PROL1 BKLHD2 ZNF204 FLJ21069 LOC147463 MLAT4 BLAME AD024 HCAP-G MGC2376 MGC3265 SCDGF-B DKFZP434F0 318 MGC13057

NM_021225 AB037730 AF033199 AK023505 AK025953 AK027294 AK057678 AK057782 BC015907 NM_018192 NM_020125 NM_020675 NM_022346 NM_023930 NM_024028 NM_025208 NM_030817 NM_032321

Proline-rich 1 KIAA1309 protein Zinc finger protein 204 Homo sapiens cDNA FLJ13443 fis，clone PLACE1002853 Homo sapiens cDNA：FLJ22300 fis，clone HRC04759 Homo sapiens cDNA FLJ14388 fis，clone HEMBA1002716 Homo sapiens cDNA FLJ33116 fis，clone TRACH2001339 Homo sapiens cDNA FLJ25053 fis，clone CBL04266 Homo sapiens，clone IMAGE：3922927，mRNA Hypothetical protein FLJ10718 BCM-like membrane protein precursor AD024 protein Chromosomecondensation protein G Hypothetical protein MGC2376 Hypothetical protein MGC3265 Spinal cord-derived growth factor-B Hypothetical protein DKFZp434F0318 Hypothetical protein MGC13057

Claims (4)

1. one group of mammary cancer marker gene group is characterized by in this group gene group one, combination a plurality of or full gene and can be used to differentiate that mammary gland is good, malignant tumour, and the title of 120 genes, sequence number and major function are described and seen Table 1 in the gene group.

2. the combined preparation of a plurality of or full gene of the described gene of claim 1 becomes that gene chip is used for very, the evaluation of malignant breast tumor, it is characterized in that the cDNA or the oligonucleotide probe of these genes are fixed into microarray, wherein each gene probe can be hybridized with the cDNA that derives from corresponding gene in the tissue samples or its amplified production specifically, by classification, microarray can be flat board, microballon, fine needle or film phase array, can be solid phase, liquid phase, can be oligonucleotide, polynucleotide or cDNA array.

3. the single or multiple genes of the described gene of claim 1 detect mRNA expression amount in the tissue sample as the goal gene of the probe of making nucleic acid molecular hybridization or RT-PCR amplification, identify good, malignant tumour according to the mRNA expression level, making nucleic acid molecular hybridization can be hybridization on solid support of the cDNA of the RNA that extracts and reverse transcription thereof, also can be the in situ hybridization of tissue slice that RT-PCR can be quantivative approach, semi-quantitative method, qualitative method.

4. utilize the described gene chip of claim 2 to differentiate mammary cancer and healthy tissues or carcinoid method:

(1) behind the cDNA of breast tumor tissues or healthy tissues sample or its amplified production fluorescent mark with comprise the gene chip hybridization of a plurality of or full gene of mammary cancer marker gene group, based on the fluorescence intensity level of each gene, utilizing non-supervision clustering method promptly to obtain can be with the gene expression profile of breast cancer tissue and the accurate classification of healthy tissues;

(2) use gene expression atlas that breast tumor is carried out differential diagnosis, may further comprise the steps: may further comprise the steps:

A) from breast tumor patient's excision sample or needle biopsy sample, extract total RNA and reverse transcription is cDNA;

B) cDNA or its amplified production to sample carries out fluorescent mark;

C) with sample behind the mark and the gene chip hybridization described in the claim 2

D), relatively patient tissue sample good, pernicious made evaluation with the gene mapping described in the claim 4 with non-supervision cluster analysis sample gene expression profile.

Images