KR20070088979A - Marker protein for diagnosis of a cancer, diagnosing method and kit for cancer using the same - Google Patents

Abstract

A cancer marker polypeptide, a method for diagnosing cancer using the same and a cancer diagnosis kit are provided to diagnose the cancer rapidly, accurately and efficiently in early stage thereof by analyzing cancer tissue or a cancer marker polypeptide in blood, thereby being usefully used for the cancer diagnosis. The cancer marker polypeptide is selected from a protein coded by polynucleotide sequences described No.1- 47. The method for diagnosing cancer comprises a step of analyzing a cancer marker polypeptide in a sample using the cancer marker polypeptide which is at least one selected from the protein coded by the polynucleotide sequences described No.1- 47. The cancer diagnosis kit comprises the cancer marker polypeptide as an antigen and at least one antibody having the specificity to the cancer marker polypeptide.

Description

Marker protein for cancer diagnosis, cancer diagnosis method and cancer diagnostic kit using the same {MARKER PROTEIN FOR DIAGNOSIS OF A CANCER, DIAGNOSING METHOD AND KIT FOR CANCER USING THE SAME}

Figure 1 shows the lymph node metastasis (Lymph node (LN) metastasis) state using the oligonucleotide microarray according to the partial least square analysis method.

Figure 2 shows a schematic diagram of a method for screening differences in the expression patterns of genes among lung cancer patients using a cDNA microarray.

Figures 3a to 3c is a schematic diagram showing a conventional manufacturing process of the antibody microarray.

4 is a schematic diagram showing a conventionally known Western blotting analysis procedure. (Source: http://www.bseinquiry.gov.uk/report/volume2/fig1_8.htm)

5 is a result of analyzing a protein specifically expressed in lung cancer tissue using the antibody microarray according to Example 2 of the present invention.

Figures 6a to 6b is a result of analyzing the protein specifically expressed in the lung cancer tissue in serum using Western blotting according to Example 3.

The present invention relates to a protein differentially expressed in a lung cancer patient, a polynucleotide encoding the same, and a use thereof, which enables early diagnosis of lung cancer by blood content, and the use of a micro array for early diagnosis of lung cancer. The reading of the results is quick and accurate and efficient, and since the diagnosis can be made only with serum, the diagnosis is easy and convenient.

Numerous cellular proteins appear to increase or decrease in the body fluids of various cancer patients. In cancer patients, it can be used for diagnosis and prognosis of cancer by analyzing the presence or absence of expression of the protein showing the cancer cell specific expression pattern. For example, an increase in prostate specific antigen (PSA) at serum levels has been used as a marker for the presence of human prostate cancer.

Several diagnostic markers that can be analyzed in blood, tissue samples, etc. are known, for example, Carcinoembryonicc antigen (CEA), alpha Fetoprotein (AFP), and prostate specific antigen (Prostate Specific Antien PSA). However, these markers have low sensitivity and specificity for the presence or absence of cancer. Therefore, the diagnosis method needs to be verified by a clinical analysis method that requires time and labor (eg, X-rays, mammograms, biopsies, etc.).

Lung cancer has increased rapidly in recent decades, and today it is the most common malignant tumor in both men and women in the West, and is the second most common malignant tumor in Korea. The frequency is surprisingly increasing in women. Symptoms of patients with lung cancer may vary from the case of accidentally detected during the physical examination or other disease in the hospital without the symptoms, depending on the pattern of lung cancer. Non-small cell lung cancer includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma except small cell lung cancer. The three tissue types have similar progression and treatment methods are classified into one. T3N0 refers to any size carcinoma invading the chest wall, diaphragm, mediastinal pleural pleural wall, pericardial pericardium, or invading the main bronchus within 2 cm of the trachea, or having a diffuse lung or obstructive lung cancer (T3). There is no local lymph node metastasis (N0). The stage of lung cancer refers to the case where the cancer is confined to the lung parenchyma and is less than or equal to 3 cm. In stage 2, the cancer is spread to the chest wall and diaphragm when the cancer is spread to the surrounding lymph nodes and to the pleura. The spread of mediastinal lymph nodes is called 3A stage, and surgery is possible up to stage 3A. 3B stage is inoperable because of invasion of blood vessels, esophagus and trachea of mediastinum. Stage 4 refers to the spread of cancer to other organs (liver, bone, brain, bone marrow).

Chest X-ray, which is the most important and convenient test for the diagnosis of lung cancer, can detect the mass of lungs. Elevation can be seen and metastases to rib cage such as ribs can be seen. In addition, computerized tomography or magnetic resonance imaging is useful for identifying the extent of involvement in the mediastinum or surrounding tissue of a primary tumor and for distant metastasis. Lung cancer can be confirmed histologically by sputum cytology, bronchoscope, and bronchial biopsy.

However, about half of lung cancer patients are already inoperable at the time of discovery, and it is determined that surgery is possible, and even a large number of them are not completely resected. This is because the progress is impossible to cure. Therefore, the early diagnosis treatment of lung cancer is the most important to increase the cure rate of lung cancer.

Lung cancer has a problem that detailed diagnosis is difficult. Even experienced veteran scientists may find it difficult to see mixed opinions about the specific types of lung cancer. Such a detailed diagnosis of lung cancer is difficult because the markers useful for the diagnosis are limited. For example, in the case of blood cancers such as leukemia or lymphoma, multiple markers are identified, making detailed diagnosis easier, whereas in the case of lung cancer. Similar problems with lung cancer are found in most solid tumors.

Therefore, there is an urgent need for the development of cancer specific markers present in biological samples including blood and methods for diagnosing cancer with high accuracy and precision using these markers.

In order to solve the problems of the prior art as described above, an object of the present invention is to provide a protein for the early diagnosis of lung cancer using a protein expression pattern of the serum of lung cancer patients and genes encoding the same.

It is another object of the present invention to provide a DNA microarray using a gene encoding a protein specifically expressed in the serum of lung cancer patients as a probe, which can increase the speed and accuracy in reading the early diagnosis of lung cancer.

Still another object of the present invention is to provide a marker for diagnosing lung cancer of proteins present in serum, and to provide a method for accurately and easily diagnosing lung cancer using only serum.

Still another object of the present invention is to provide a method for diagnosing cancer using a protein specifically expressed in the serum of a lung cancer patient or a gene encoding the protein as a marker for diagnosing lung cancer.

It is another object of the present invention to provide an antibody against the protein, and a method for accurately and easily diagnosing lung cancer using the same.

The present invention is a lung cancer marker polypeptide, and lung cancer diagnostic method using the same, an antibody against the polypeptide (polypeptide), and a method for producing the antibody, for lung cancer diagnostics comprising a base sequence corresponding to the lung cancer marker polypeptide It relates to nucleic acid probes.

In addition, the present invention relates to a lung cancer marker polypeptide consisting of a protein encoded by a polynucleotide sequence shown in Tables 1A and 1B, and a gene encoding the same.

To provide a lung cancer diagnostic kit (kit) comprising at least one antibody having a specificity.

TABLE 1a

No GB Accession Gene Symbol Gene Name Regulation (p ositive _nega tive ) One NM_000668 ADH1B alcohol dehydrogenase IB (class I), beta polypeptide DOWN 2 NM_020533 MCOLN1 mucolipin 1 DOWN 3 NM_022145 null null UP 4 NM_001823 CKB creatine kinase, brain DOWN 5 NM_057090 ARTN artemin DOWN 6 NM_004510 SP110 SP110 nuclear body protein DOWN 7 NM_012138 AATF apoptosis antagonizing transcription factor UP 8 NM_002262 KLRD1 killer cell lectin-like receptor subfamily D, member 1 DOWN 9 NM_021727 FADS3 fatty acid desaturase 3 DOWN 10 NM_003975 SH2D2A SH2 domain protein 2A DOWN 11 NM_000080 CHRNE cholinergic receptor, nicotinic, epsilon polypeptide DOWN 12 NM_000992 RPL29 ribosomal protein L29 UP 13 NM_021809 TGIF2 TGFB-induced factor 2 (TALE family homeobox) DOWN 14 NM_001246 ENTPD2 ectonucleoside triphosphate diphosphohydrolase 2 DOWN 15 NM_016153 null null DOWN 16 NM_007063 TBC1D8 TBC1 domain family, member 8 (with GRAM domain) DOWN 17 NM_000191 HMGCL 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase (hydroxymethylglutaricaciduria) DOWN 18 AC022003 null null DOWN 19 NM_014621 HOXD4 homeo box D4 DOWN 20 NM_015909 null null DOWN 21 NM_003752 EIF3S8 eukaryotic translation initiation factor 3, subunit 8, 110 kDa UP 22 NM_001651 AQP5 aquaporin 5 DOWN 23 NM_014629 ARHGEF10 Rho guanine nucleotide exchange factor (GEF) 10 DOWN

TABLE 1b

No GB Accession Gene Symbol Gene Name Regulation (pos itive _negative) 24 NM_001915 CYB561 cytochrome b-561 DOWN 25 NM_016283 TAF9 TAF9 RNA polymerase II, TATA box binding protein (TBP) -associated factor, 32kDa DOWN 26 NM_001448 GPC4 glypican 4 DOWN 27 NM_023000 ARID4A AT rich interactive domain 4A (RBP1-like) DOWN 28 NM_003213 TEAD4 TEA domain family member 4 UP 29 NM_004224 GPR50 G protein-coupled receptor 50 DOWN 30 NM_005798 RFP2 ret finger protein 2 DOWN 31 NM_022338 C11orf24 chromosome 11 open reading frame 24 UP 32 ENSG00000100365 null null DOWN 33 AF123073 null null DOWN 34 AF151019 null null UP 35 NM_015939 null null DOWN 36 NM_002388 MCM3 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) UP 37 ENSG00000084774 null null UP 38 AF216972 null null DOWN 39 XM_041585 null null DOWN 40 AL022312 null null DOWN 41 M98331 DEFA6 defensin, alpha 6, Paneth cell-specific DOWN 42 AC005754 null null DOWN 43 NM_003093 SNRPC small nuclear ribonucleoprotein polypeptide C UP 44 AC026487 null null DOWN 45 NM_004639 BAT3 HLA-B associated transcript 3 UP 46 AF100318 MAP3K6 mitogen-activated protein kinase kinase kinase 6 UP 47 AF348074 null null DOWN

The present invention also provides a method for diagnosing cancer, which comprises analyzing a cancer marker polypeptide contained in a sample with one or more cancer marker polypeptides selected from the group consisting of proteins encoded by the polynucleotide sequences shown in Tables 1A and 1B. It is about.

The present invention relates to an antibody having a cancer marker polypeptide selected from the group consisting of proteins encoded by the polynucleotide sequences shown in Tables 1A and 1B as an antigen and having specificity for the cancer marker polypeptide.

The present invention is prepared by using a cancer marker polypeptide selected from the group consisting of proteins encoded by the polynucleotide sequences shown in Tables 1A and 1B as antigens and containing at least one antibody having specificity for the cancer marker polypeptide. It relates to a diagnostic kit.

The present invention relates to a polynucleotide probe for diagnosing cancer, comprising the polynucleotide sequence shown in Tables 1a and 1b above and a polynucleotide sequence complementary to the polynucleotide sequence. The present invention also relates to a DNA microarray of the polynucleotide probe.

Hereinafter, the present invention will be described in more detail.

The inventors were able to find polypeptides used for early diagnosis of lung cancer, antibodies and diagnostic kits using the polypeptides in the blood content of 47 serum proteins which are differentially expressed in lung cancer patients. Accordingly, the present invention provides a lung cancer marker polypeptide comprising an amino acid sequence encoding a protein encoded by the polynucleotide sequences shown in Tables 1a and 1b. The present invention also provides a polypeptide fragment that induces antibody production against the lung cancer marker polypeptide.

The present invention provides a lung cancer diagnostic kit containing at least one antibody having specificity to a lung cancer marker polypeptide including a protein encoded by the polynucleotide sequences shown in Tables 1A and 1B.

In the present specification, the cancer marker polypeptide is more specifically a change in expression level in the cells obtained from a lung cancer patient or in the blood of a lung cancer patient. In addition, the antibodies of the present specification are polyclonal antibodies or monoclonal antibodies prepared by using a polypeptide comprising a protein encoded by the polynucleotide sequences shown in Tables 1A and 1B and specific for the marker polypeptide.

Cancer marker polypeptides selected from the group consisting of proteins encoded by the polynucleotide sequences shown in Tables 1a and 1b of the present invention are known to have specific functions or organs that express them, respectively. Until now, the relationship with lung cancer is not fully known. Thus, the inventors have found that such polypeptides can be used as lung cancer markers.

In the present invention, the antibody can be prepared by a generally known method using the polypeptide. The antibody of the present invention is an antibody having specificity for a lung cancer marker polypeptide, including a protein encoded by the polynucleotide sequences shown in Tables 1A and 1B, and may be either a polyclonal antibody or a monoclonal antibody. As a method for analyzing the cancer marker polypeptide contained in a sample, for example, lung cancer tissue, blood of a cancer patient or serum using the antibody of the present invention, a conventional antigen-antibody reaction assay can be used. For example, the immunoassay (measuring) method may include enzyme immunoassay (ELISA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay, immunoblot, western blot, Immunostaining;

Western blot method is an effective method for determining the molecular weight of the protein in the biological sample. For example, biological sample liquids, such as extracts derived from organ tissues, are subjected to acrylamide gel electrophoresis, and then transferred to a membrane, reacted with an antibody that recognizes the protein or the peptide, and generates an immune complex. Labeled second antibody is used and detected.

Immunostaining is an effective method for analyzing the site of expression of a polypeptide of interest in tissues and cells, for example, by reacting and producing tissue fragments, cells, etc. immobilized on slide glass with the antibodies of the present invention. Immune complexes are performed by using a labeled second antibody and detecting it.

An ELISA method is mentioned as a preferable example of the immunoassay using the antibody which recognizes the said lung cancer marker polypeptide. The ELISA method can be carried out according to methods such as a general competition method and a sandwich method, and can also be carried out in a liquid or solid state system.

Cancer diagnosis kit (kit) of the present invention comprises the above polyclonal or monoclonal antibodies, and more specifically, it comprises at least one amino acid sequence encoding the protein shown in Table 1a and Table 1b By using at least 1 type or more of antibodies which have specificity for a marker polypeptide, it can be used for diagnosis of lung cancer.

In addition, the diagnostic kit may use an immune response as necessary, and may include a well, a stain, an enzyme-labeled antibody for detection, a washing solution, an antibody diluent, a sample diluent, an enzyme substrate, an enzyme substrate diluent, and other reagents. .

The present invention will be described in more detail with reference to the following illustrative examples, but the protection scope of the present invention is not limited to the following examples.

EXAMPLE

Example  1: using nucleic acid micro array mRNA  Aspect analysis

A. Sample Preparation

A-1: Lung Cancer Tissue

Samples of 120 lung cancer tissues were collected from non-small cell lung cancer (NSCLC) patients who underwent pneumonectomy at Samsung Seoul Hospital from 1995 to December 2001. Table 2 below summarizes the clinical information of 112 non-small cell lung cancer patients.

Tissues were obtained after thoracotomy from a T3N0 stage patient. Of the 112 lung cancer tissues, 70 are LN (-) and 42 are LN (+). Histologic types of sample tissue were 45 adenocarcinomas, 58 squamous cell carcinomas, and 6 large cell carcinomas. 46 patients recurred. (Patients with incomplete resection or no mediastinal resection are excluded.)

TABLE 2

Clinical Information of 112 Patients with Non-Small Cell Lung Cancer

Total 112 samples: LN (+) 42 samples, LN (-) 70 samples

   (Excluding 15 samples of T3N0 and T4N0 of 127 samples)

Stage: 70 stages I, 14 samples II, 28 samples III

Gender: 82 samples for men, 30 samples for women

Age: 13 ~ 80 years old (median = 62 years old)

Cell Type: ADC 46 samples, BAC 2 samples, SQC 58 samples, LAC 6 samples

Size: 1.2 ~ 19 cm (median = 4 cm)

OP date: 1995.04.21 ~ 2002.06.03 (median: 1999.05.30)

Smoking capacity: 77 smokers, 33 non-smokers, 2 samples unknown

A-2: Tissue Sample Preparation and RNA Extraction

After routine mediastinal examination, incision of all lymph nodes was performed systematically by thoracotomy. All lymph nodes removed 11, 10, 9, 8, 7, 4, 3, 2 parts of the right lung cancer tissue and 11, 10, 9, 8, 7, 6, 5 parts of the American Thoracic Society LN map. Cut off 6 (range: 5-8) parts of the mediastinum on average. All surgically removed tissues were immediately examined pathologically. And avoiding the necrotic part of the rock mass, one to two pieces (5mm × 5mm) of the surrounding cancer tissue was removed and stored at -80 ° C. In looking at the medical data, hematoxylin and eosin slides in lymph node tissues were reviewed by a confirmed pathologist. 2188 lymph nodes were removed from all patients (the number of LNs per patient: 21.3 ± 10.6, range: 8-54), and 152 lymph nodes were positive for metastasis.

112 lung cancer tissues microdissected 112 cancer tissues from 112 patients. And all cancer tissues were lightly stained with hematoxylin to see well before RNA extraction. Each micro-incisional tissue consists of more than 90% of cancer cells.

1 mL of Trizol reagent (Life Technologies, Rockville, MD) was added to the dissected cancer tissues and immediately vortexed and homogenized. Total RNA was isolated according to Trizol reagent protocol and quantitated total RNA was analyzed by electrophoresis on 1% agarose gel containing 0.6 M formaldehyde and ethidium bromide. Quantification of total RNA was analyzed using a Nanodrop spectrometer (Nanodrop Technologies, Rockland, DE).

B. In non-small cell lung cancer  Location and scope before lymph nodes

According to the 1997 TNM classification (a table of pathologically classified prognosis after lung cancer resection, oncology, oncology = Oncology, Park Jae-gap; Park Chan-il; Kim, No-Kyung, Sculpture, 2003.) Ten patients (63%) had a pN0 stage, 42 patients (38%) had metastasized to lymphatic vessels, 19 patients (17%) had a pN1 stage and 23 patients (21%) had a pN2 stage. More than 2000 nodes ( n = 2349) with an average of 21 lymph nodes (8-54) were analyzed clinically. 148 lymph nodes (6.3%) were metastatically related, while 2201 LNs (93.7%) were cancer free. LN involvement was seen in 44% of patients with squamous cell carcinoma and in 33% of patients with large cell carcinoma. Patients with adenocarcinoma have an LN involvement rate of 29%.

For reference, T and N lesions are factors that affect survival after resection of lung cancer, and lymph node metastasis. The number of metastasized lymph nodes and the number of metastasized lymph nodes are all related to prognosis. On the other hand, the type of carcinoma, metastasized lymph node area, and the metastasis of the metastasized lymph node N1 do not affect survival.

C. Using cDNA Microarrays mRNA  Expression Analysis

About 5-10 ug of total RNA isolated from cancer tissue was used for oligonucleotide microarray analysis (Macrogen, Seoul, Korea), and the remainder was amplified by RT-PCR or real-time PCR. Oligo Human 10K microarray from Macrogen spotted 10,416 human genes, known 8032 genes and 2076 expressed sequence tags (ESTs) genes with 50-mer oligonucleotide probes. Each of the 112 total RNAs was analyzed and confirmed by hybridization with a universal human reference RNA (stratagene, La. Jolla, Calif.) And scanning (oligonucleotide microarray hybridization).

Microarray hybridized images were scanned and analyzed using Imagen version 5.5 software (BioDiscovery, El Segundo, Calif.). The data were then generalized by the lowess method. In the lowess method, the ratio of Cy-3 to Cy-5 is basically converted to log.

Partial least square (PLS) analysis was carried out to investigate whether the expression of genes in cancer tissues could be clearly distinguished according to the presence or absence of metastasis to the lymph nodes in lung cancer patients. Was performed. And. Permutation t test was performed by multiple testing adjustment (MTA) including Westfall-Young method to select genes with different expression patterns.

Oligonucleotide microarray experiments revealed the expression of mRNA from lung cancer patients with or without metastasis.

Partial least square analysis was used to identify differences in gene expression patterns among patients with or without lung cancer. The results are shown in Figure 1, showing that the LN metastasis status is well distinguished, it is possible to select a group of genes associated with LN metastasis. In addition, Figure 2 shows a schematic diagram of a method for screening the difference in expression patterns of genes between lung cancer patients, using a cDNA micro array.

D. Development of classification models and the accuracy of their predictions

Next, two classification models were developed using two model gene sets. Each of these two types is characterized. Through this modeling process, we designed an integrated and modified algorithm to increase the accuracy of prediction in a given model.

In order to increase the accuracy of lymph node metastasis prediction, modified algorithms were developed by incorporating methods adopted by algorithms such as vector machines, artificial neural networks, decision trees, and naive bayes. Three of these algorithms show an acceptable level of prediction accuracy of over 80% and incorporate three algorithms adopted in the modified algorithm. In the modified algorithm, the lymph node metastasis state was determined to be positive or negative, and two to three algorithms were combined and applied. This modified algorithm was applied to select 47 genes and generalize the classification model. In addition, by 10-fold cross validation, Model A shows 95.24% sensitivity and 90.00% specificity, which is higher than any other algorithm (Table 3).

TABLE 3

Behavior of Models A and B in Four Classification Algorithms

According to the above method. The accuracy of the prediction was tested by applying the model obtained from 10-fold cross-validation and the patient's training set and test set. To support the usefulness of the classification model for clinical recurrence, we analyzed Kaplan-Meier survival curves using the long-rak test to identify distinct patterns in lymph node metastasis.

Based on the results of item B of Example 1, an attempt was made to distinguish genes differentially expressed in 70 patients who did not have lymph node metastasis and 42 patients who had lymph node metastasis. (A) Samples from all 112 patients were cross-validated and classified as model A. Statistically transformed t-tests were performed by adjusting P -values with multiple testing using the Westfall-Young method to select model gene sets.

Model gene set A consists of 47 genes ( P <0.05). Thirty-nine known genes, eight ESTs, and their features are summarized in Tables 4 and 5.

TABLE 4

Genes representing differences in expression of mRNA expression patterns of 112 patients with non-small cell lung cancer (Model gene set A)

Gene Accession no. Gene description Gene Symbol AC005754 EST AC022003 EST AC026487 EST AF100318 mitogen-activated protein kinase kinase kinase 6 MAP3K6 AF123073 LOC81558 LOC81558 AF151019 Hypothetical protein HSPC111 HSPC111 AF216972 EST AF348074 n-acetyltransferase 2 NAT2 AL022312 EST ENSG00000084774 EST ENSG00000100365 EST M98331 defensin, alpha 6, Paneth cell-specific DEFA6 NM_000080 cholinergic receptor, nicotinic, epsilon polypeptide CHRNE NM_000191 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase (hydroxymethylglutaricaciduria) HMGCL NM_000668 alcohol dehydrogenase IB (class I), beta polypeptide ADH1B NM_000992 ribosomal protein L29 RPL29 NM_001246 ectonucleoside triphosphate diphosphohydrolase 2 ENTPD2 NM_001448 glypican 4 GPC4 NM_001651 aquaporin 5 AQP5 NM_001823 creatine kinase, brain CKB NM_001915 cytochrome b-561 CYB561 NM_002262 killer cell lectin-like receptor subfamily D, member 1 KLRD1 NM_002388 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) MCM3 NM_003093 small nuclear ribonucleoprotein polypeptide C SNRPC NM_003213 TEA domain family member 4 TEAD4 NM_003752 eukaryotic translation initiation factor 3, subunit 8, 110 kDa EIF3S8 NM_003975 SH2 domain protein 2A SH2D2A NM_004224 G protein-coupled receptor 50 GPR50 NM_004510 SP110 nuclear body protein SP110 NM_004639 HLA-B associated transcript 3 BAT3 NM_005798 ret finger protein 2 RFP2 NM_007063 TBC1 domain family, member 8 (with GRAM domain) TBC1D8 NM_012138 apoptosis antagonizing transcription factor AATF NM_014621 homeo box D4 HOXD4 NM_014629 Rho guanine nucleotide exchange factor (GEF) 10 ARHGEF10 NM_015909 Neuroblastoma-amplified protein NAG NM_015939 CGI-09 protein CGI-09 NM_016153 LW-1 LW-1 NM_016283 TAF9 RNA polymerase II, TATA box binding protein (TBP) -associated factor, 32kDa TAF9 NM_020533 mucolipin 1 MCOLN1 NM_021727 fatty acid desaturase 3 FADS3 NM_021809 TGFB-induced factor 2 (TALE family homeobox) TGIF2 NM_022145 Leucine zipper protein FKSG14 FKSG14 NM_022338 chromosome 11 open reading frame 24 C11orf24 NM_023000 AT rich interactive domain 4A (RBP1-like) ARID4A NM_057090 artemin ARTN  XM_041585 EST

TABLE 5

Genes representing differences in expression of mRNA expression patterns of 112 patients with non-small cell lung cancer (Model gene set A)

Gene representing expression difference from mRNA expression analysis of 79 patients with non-small cell lung cancer (Model gene set B)

Gene Accession no. Gene description Gene Symbol AC005754 EST AF216972 EST AF309034 SRY (sex determining region Y) -box 6 SOX6 ENSG00000084774 EST NM_000486 aquaporin 2 (collecting duct) AQP2 NM_000992 ribosomal protein L29 RPL29 NM_001246 ectonucleoside triphosphate diphosphohydrolase 2 ENTPD2 NM_001448 glypican 4 GPC4 NM_001915 cytochrome b-561 CYB561 NM_002262 killer cell lectin-like receptor subfamily D, member 1 KLRD1 NM_002280 keratin, hair, acidic, 5 KRTHA5 NM_002388 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) MCM3 NM_003093 small nuclear ribonucleoprotein polypeptide C SNRPC NM_003365 ubiquinol-cytochrome c reductase core protein I UQCRC1 NM_003481 ubiquitin specific protease 5 (isopeptidase T) USP5 NM_003975 SH2 domain protein 2A SH2D2A NM_004639 HLA-B associated transcript 3 BAT3 NM_004711 synaptogyrin 1 SYNGR1 NM_012138 apoptosis antagonizing transcription factor AATF NM_014621 homeo box D4 HOXD4 NM_014629 Rho guanine nucleotide exchange factor (GEF) 10 ARHGEF10 NM_015909 Neuroblastoma-amplified protein NAG NM_016153 LW-1 LW-1 NM_018695 erbb2 interacting protein ERBB2IP NM_020533 mucolipin 1 MCOLN1 NM_021809 TGFB-induced factor 2 (TALE family homeobox) TGIF2 NM_022338 chromosome 11 open reading frame 24 C11orf24 NM_030673 SEC13-like 1 (S. cerevisiae) SEC13L1 NM_057090 artemin ARTN XM_039877 mucin 5, subtype B, tracheobronchial MUC5B XM_041585 EST

Example  2: Analysis with Antibody Microarrays

2-1: Preparation of Antibodies and Serums

Thirteen antibodies were selected based on the results of the DNA microarrays (Tables 4 and 5), and serum amyloid A (SAA) was selected by reference search. Anti-aquaporin 5 (AQP5), anti-creatine kinase brain (CKB), anti-homeobox protein (HESX1), anti-minochromosome maintenance protein (MCM3), anti-TATA-binding protein (TFIID) and anti-5'-TG -3 'interacting protein (TGIF) is derived from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-apoptosis antagonizing transcription factor (AATF) and anti-SoxLZ / Sox6-binding protein (FKSG14) are available from Abnova Corporation (Taipei, Taiwan), anti-eukaryotic translation initiation factor 3, subunit 8 (eIF3S8) from Bethyl Laboratories (Montgomery, TX, USA), and anti-small nuclear ribonucleoprotein polypeptide C (SNRPC) from GeneTex (San Antonio, TX, USA). From anti-mitogen-activated protein kinase kinase kinase 6 (MAP3K6) from Abgent (San Diego, CA, USA), Anti-TEA domain family member (TEAD4) from Abcam (Cambridge, UK) and Anti-SAA from Antigenix Each was purchased from America (Huntington Station, NY, USA) and used (Table 6).

TABLE 6

Target Antibodies Used in Antibody Microarrays (1 to 13) and Western Blotting (1 to 6)

Serum was collected in 7 ~ 10ml of blood of normal group, lung cancer malignant tumor group of Samsung Seoul Hospital, benign tumor patient group, centrifuged at 3000rpm for 10 min at 4 ° C, and separated in the same way. Store at 80 ° C.

A total of 25 serum samples were used in the present experiment, 8 from normal patients, 4 from lung cancer patients of Samsung Medical Center, and 4 from benign tumor patients. All serum was stored frozen until analysis.

2-2: Fluorescent Labeling of Serum

Serum was labeled with Cy3 and Cy5 for comparative analysis of the expression patterns of two patient group proteins in a microarray. As shown in Figure 3a, the serum was labeled with fluorescence by reacting with a serum with N-hydroxysuccinimide ester (NHS) -linked Cy3 and Cy5 dye (Amersham Biosciences, Buckinghamshire, UK). First, dilute the serum to 1/15 with dye-conjugation buffer (50 mM sodium carbonate buffer, pH 9.0) and gently shake it every 10 minutes in the dark to mix the Cy dye in the dye-conjugation buffer with the diluted serum. Reaction was performed at room temperature for 40 minutes while giving. 1 M Tris-HCl (pH 8.0) is added and the reaction of serum and Cy dye is stopped for 20 minutes with shaking every 10 minutes in the dark. Only fluorescently labeled serum was isolated by centrifugation at 10,000 × g for 30 minutes using Microcon YM-10 (Millipore Corporate, Billerica, Mass., USA). To stabilize the fluorescently labeled serum, PBS containing skim milk of about 50 times its volume was added and centrifuged again to obtain only fluorescently stable serum. (Finally, the fluorescently labeled serum was about 15 times in PBS. Diluted.)

2-3. Antibody Microarray Fabrication

Antibody microarrays were hydrogel coated glass slides (slides H Schott Nexterion, Jena) with CMP 3 spotting pins (Telechem International, Sunnyvale, CA, USA) using a robotic arrayer (Microsys, Cartesian Technologies, Irvine, CA, USA). , Germany) and the preparation method is shown in Figure 3b.

 One antibody microarray consists of 13 antibody sets in Table 6, the double positive control being anti-human serum albumin (Sigma-Aldrich, St. Louis, MO, USA) and the negative control being anti Bovine serum albumin (anti-BSA, Sigma-Aldrich). All protein solutions required for the spot were prepared by mixing 0.5% trehalose, and the relative humidity of the spot machine was maintained at 70%. Each Ab sample was spotted 4 rows per array. Allow the antibody to covalently bond with the slide reactor at room temperature for 1 hour, then remove the slide out of the array and react overnight at 4 ° C with phosphate buffer (PBS) containing 1M ethanolamine (Sigma-Aldrich). And stopped. After washing with PBS, the prepared antibody microarray was reacted with fluorescently labeled serum samples for 1 hour in the dark. Before confirming the fluorescence intensity, the antibody microarray reacted with the fluorescent labeled serum sample was washed three times successively with PBST (0.1% Tween-20 containing PBS), washed with distilled water, and dried with N2 gas.

2-4. Analysis of Fluorescence Intensity

Using a fluorescence scanner (GenePix Personal 4100A; Axon instruments, Union City, CA, USA), the fluorescence intensities emitted by specific wavelengths of Cy5- and Cy3- are read and scanned using the GenePix Pro 4.1 software provided by the manufacturer. The data were analyzed. The photomultiplier tube (PMT) values of the two channels are adjusted so that the Cy5 / Cy3 ratio of the microarray is one. The intensity of Cy5 and Cy3 from all spots was calculated by subtracting the median background intensity. Four calibrated data for each antibody were used to obtain an average value for future analysis. Spots with obvious defects or no signal more than twice the background intensity were excluded from the analysis. The ratio of protein expression between the two samples is expressed as internally normalized ratio (INR). This ratio was calculated using values obtained by differently labeling the two types of samples with the two dyes. The first slide was labeled with a Cy3-labeled reference and a Cy5-sample and bound to the antibody microarray. The second slide was labeled with a Cy5-standard sample and a Cy3-test sample to bind the same amount to the antibody microarray, respectively. Cy5- and Cy-3 labeled antibodies are shown in Figure 3c.

 The ratio of Cy5 and Cy3 is obtained on each of the two slides, and then the internally normalized ratio (INR) is calculated as follows.

[Equation 1]

 Ideally, R1 and R2 will be the same. Therefore INR is defined as the square root of (X / Y). INR is not affected by dye-labeling efficiency and can be used as a general condition for all proteins without external normalization requirements. Here, all ratios are converted to log2 and displayed as convention.

2-4: Experimental Results

Serum of lung cancer patients shown in Figure 4, and the serum of healthy, benign tumor patients were analyzed using an antibody microarray. Serum consisting of four normal sera was used as a reference.

When analyzing sera from lung cancer patients and positive patients, spots consisting of antibodies of only 8 proteins of AQP5, CKB, HESX1, MCM3, TFIID, TGIF, AATF, and SAA show high fluorescence intensity on both slides. 4A, 4C). Serum from normal subjects showed the same intensity except for antibody SAA (FIG. 4B). However, in all cases, no signal was seen in antibody spots of eIF3S8, FKSG14, TEAD4, MAP3K6, and TEAD4 proteins. This result is thought to be because the antibody microarray was not performed on mRNA to lung tissue, unlike the results of DNA microarray, but on serum in which a large amount of protein is present. Although proteins transcribed in mRNA are expressed at very high levels in lung tissues, homologous proteins may not be present at the same level in lungs as in lung tissues. In addition to the differences in the tissues, the actual protein expression level is thought to be the result of showing little correlation with the mRNA expression level.

The background intensity of the slide appears sufficiently lower than the fluorescence intensity of the spot. And the shape of the spot does not deviate from the normal shape of the circle. Spot diameters vary with the concentration of antigen and antibody and the conditions of the buffer. However, if these spots do not overlap or appear evenly with other surrounding spots, the fluorescence signal is not affected by the diameter of the spot. From all these facts, it was confirmed that protein microarrays using the currently available antibodies can be used to analyze expression patterns of human serum proteins by fluorescence of the serum with two kinds of dyes.

Expression analysis of proteins, Relative proteins of the three test sample groups were calculated by conversion to INR and log2 values (FIG. 4A). The log2 (INR) value of a particular protein is higher than 1.0 or greater than 1.0, meaning that it is up-regulated and down-regulated, respectively. As expected, there was no difference between the expression rates of normal and serum samples. Since the standard sample is a collection of four normal human serum, these results indicate that the standard sample worked properly for profiling in the antibody microarray. In contrast, serum from lung cancer patients showed a significant difference in expression when compared to standard samples, with the exception of HAS and AATF. Contrary to the results of normal human expression, AQP5, CKB, HESX1, MCM3, TFIID, and TGIF showed lower levels of expression in lung cancer patients with six proteins than in normal human serum. . Unlike the other antibodies selected, HAS (human serum albumin) on the right side of the table was used as a positive control, not a candidate protein of the biomarker, so it was confirmed that there was no difference in the expression level of the microarray. SAA was confirmed to be up-regulated in lung cancer patients as in other papers, and the same results are expected in the positive patients. If protein expression levels are statistically significant between two patient groups, the protein is still meaningful as a biomarker, even if both patients are up-regulated. Therefore, SAA is also required for experiments to confirm its availability as a biomarker by statistical analysis. Since there may be a difference in protein expression levels between lung cancer patients (malignant tumor) and benign tumor patients, it is necessary to analyze the expression patterns of the patients with benign tumors. Except for the SAA mentioned above, all of the normal serum protein expression patterns were similar. In this regard, it was confirmed that the antibody microarray can distinguish lung cancer (malignant tumor) patients from benign tumor patients as well as normal people. Furthermore, lung cancer diagnosis was possible by analyzing the difference in expression rate of these proteins.

The expression level of mRNA between lung cancer tissue and normal lung tissue analyzed by the antibody microarray is shown in the following table, in which the down-regulated mRNA is represented by a black inverted triangle (▼) and the protein is up-regulated. Denotes an empty triangular shape (Δ).

TABLE 7

MRNA quantification between lung cancer tissue and normal lung tissue analyzed by antibody microarray

Example  3: Weston Blotting

Western blotting is a method of finding only the desired protein in a protein mixture by using an antibody that reacts with an antigenic epitope. As a result of cDNA microarray analysis, expression of a protein that is thought to be differentially expressed between lung cancer patients and normal people. This was done to confirm the results of the microarrays on the aspects.

B-1. Preparation of Serum Samples

ProteoExtract Albumin / IGg Removal Kit (Calbiochem, La Jolla, Calif.) Was used to remove albumin and IGg, which make up the majority of serum proteins. (The serum is diluted about 1/30.) The diluted serum was quantified by Bradford, mixed with 2X SDS sample buffer, boiled for 3 minutes, and centrifuged at 12000 rpm for 4 minutes to collect samples at the bottom of the tube.

TABLE 8

Antibodies Used in Western Blotting

Primary antibody  Secondary antibody Primary antibody size 1st dilution range 2nd dilution range AQP5 SC-2004 (goat anti-rabbit IgG-HRP) 35kDa 1: 100 ~ 1: 1000 1: 2000 ~ 1: 100000 CK-B SC-2020 (donkey anti-goat IgG-HPR) 43kDa 1: 100 ~ 1: 1000 1: 2000 ~ 1: 100000 HESX-1 SC-2020 (donkey anti-goat IgG-HPR) 21kDa 1: 100 ~ 1: 1000 1: 2000-1: 5000 MCM3 SC-2020 (donkey anti-goat IgG-HPR) 115 kDa 1: 100 ~ 1: 1000 1: 2000 ~ 1: 100000 TFIID SC-2004 (goat anti-rabbit IgG-HRP) 36 kDa 1: 100 ~ 1: 1000 1: 2000 ~ 1: 100000 TGIF SC-2004 (goat anti-rabbit IgG-HRP) 35 kDa 1: 100 ~ 1: 1000 1: 2000-1: 5000

C-2. Weston Blotting

The prepared serum samples were electrophoresed at 70V for 15 minutes and 120V for 1 hour in 12% SDS polyamide gel and the protein was transferred to a nitrocellulose membrane for 1 hour at 300 mA. The protein-transferred membrane was blocked with TBS-T buffer (0.1% Tween-20) containing 5% skim milk at room temperature for 1 hour. Antibodies against the selected protein were diluted in TBS-T buffer at 1/2000 with the primary antibody and reacted overnight at 4 ° C. The membrane with primary antibody was washed five times every 10 minutes with TBS-T buffer (0.1% Tween-20), and the secondary antibody was diluted in TBS-T buffer at 1/5000 and bound at room temperature for 1 hour. Rinse the membrane 5 times with TBS-T buffer every 10 minutes, then react with the membrane and Western blotting reagents from Santa Cruz Biotechnology (Santa Cruz, CA, USA) for 1 minute and develop with Hyperfilm (Amersham Biosciences, Uk) in the dark. It was. Western blotting procedures are shown in FIG. 5.

Western blotting was performed for each antibody to confirm the analysis results of antibody microarrays. In this experiment, we performed Western blotting on the target proteins AQP5, CKB, HESX1, MCM3, TFIID, and TGIF in serum. In the case of AQP5, it was confirmed that the expression level of protein between lung cancer patients and normal subjects was down-regulated as in microarray results. (FIG. 6B) In FIG. 6B, Δ is Khan N, Cromer CJ, Campa M, Patz EF Jr. Clinical utility of serum amyloid A and macrophage migration inhibitory factor as serum biomarkers for the detection of nonsmall cell lung carcinoma. Cancer. The results of the ELISA experiment described in 2004 Jul 15; 101 (2): 379-84.

For five other proteins except AQP5, the desired protein was not clearly detected. In the case of antibody microarrays, even a very small amount of sample has high sensitivity for analyzing the expression of the protein, but it is difficult to detect when the amount of protein contained in the sample of Western blotting is small. In addition, since serum contains a large amount of albumin and IGg, which affects protein separation by electrophoresis, much caution is required for western blotting of serum, and Western blotting has a large amount of protein and various kinds of proteins. Serum containing is not a suitable sample. In addition, due to the dilution effect by blood, it may be difficult to detect by western blotting because the amount of target protein in proportion to the total protein of serum may be present at a very low level.

 The present invention is a patent for the early diagnosis of lung cancer in the blood content of 47 serum proteins which are differentially expressed in lung cancer patients, and when using the protein chip according to the invention, the results of the early diagnosis of lung cancer The readings are quick, accurate and efficient, and can be diagnosed with serum only, making diagnosis easy and convenient.

Claims (5)

A cancer marker polypeptide selected from the group consisting of proteins encoded by the polynucleotide sequences shown in Tables 1A and 1B below. TABLE 1a No GB Accession Gene Symbol Gene Name One NM_000668 ADH1B alcohol dehydrogenase IB (class I), beta polypeptide 2 NM_020533 MCOLN1 mucolipin 1 3 NM_022145 null null 4 NM_001823 CKB creatine kinase, brain 5 NM_057090 ARTN artemin 6 NM_004510 SP110 SP110 nuclear body protein 7 NM_012138 AATF apoptosis antagonizing transcription factor 8 NM_002262 KLRD1 killer cell lectin-like receptor subfamily D, member 1 9 NM_021727 FADS3 fatty acid desaturase 3 10 NM_003975 SH2D2A SH2 domain protein 2A 11 NM_000080 CHRNE cholinergic receptor, nicotinic, epsilon polypeptide 12 NM_000992 RPL29 ribosomal protein L29 13 NM_021809 TGIF2 TGFB-induced factor 2 (TALE family homeobox) 14 NM_001246 ENTPD2 ectonucleoside triphosphate diphosphohydrolase 2 15 NM_016153 null null 16 NM_007063 TBC1D8 TBC1 domain family, member 8 (with GRAM domain) 17 NM_000191 HMGCL 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase (hydroxymethylglutaricaciduria) 18 AC022003 null null 19 NM_014621 HOXD4 homeo box D4 20 NM_015909 null null 21 NM_003752 EIF3S8 eukaryotic translation initiation factor 3, subunit 8, 110 kDa 22 NM_001651 AQP5 aquaporin 5 23 NM_014629 ARHGEF10 Rho guanine nucleotide exchange factor (GEF) 10 TABLE 1b No GB Accession Gene Symbol Gene Name 24 NM_001915 CYB561 cytochrome b-561 25 NM_016283 TAF9 TAF9 RNA polymerase II, TATA box binding protein (TBP) -associated factor, 32kDa 26 NM_001448 GPC4 glypican 4 27 NM_023000 ARID4A AT rich interactive domain 4A (RBP1-like) 28 NM_003213 TEAD4 TEA domain family member 4 29 NM_004224 GPR50 G protein-coupled receptor 50 30 NM_005798 RFP2 ret finger protein 2 31 NM_022338 C11orf24 chromosome 11 open reading frame 24 32 ENSG00000100365 null null 33 AF123073 null null 34 AF151019 null null 35 NM_015939 null null 36 NM_002388 MCM3 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) 37 ENSG00000084774 null null 38 AF216972 null null 39 XM_041585 null null 40 AL022312 null null 41 M98331 DEFA6 defensin, alpha 6, Paneth cell-specific 42 AC005754 null null 43 NM_003093 SNRPC small nuclear ribonucleoprotein polypeptide C 44 AC026487 null null 45 NM_004639 BAT3 HLA-B associated transcript 3 46 AF100318 MAP3K6 mitogen-activated protein kinase kinase kinase 6 47 AF348074 null null A method for diagnosing cancer, characterized by analyzing a cancer marker polypeptide in a sample with one or more cancer marker polypeptides selected from the group consisting of proteins encoded by the polynucleotide sequences shown in Tables 1a and 1b of claim 1. An antibody prepared by using a cancer marker polypeptide selected from the group consisting of proteins encoded by the polynucleotide sequences shown in Tables 1a and 1b of claim 1 as an antigen and having specificity for the cancer marker polypeptide. A cancer diagnostic polypeptide prepared by using a cancer marker polypeptide selected from the group consisting of proteins encoded by the polynucleotide sequences shown in Tables 1a and 1b of claim 1 and containing one or more antibodies having specificity for the cancer marker polypeptide. Kit. A nucleic acid probe for diagnosing cancer, comprising the polynucleotide sequence shown in Tables 1a and 1b and a polynucleotide sequence complementary to the polynucleotide sequence.

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