KR20080107326A - Novel biomaker for the diagnosis of lung cancer - Google Patents

Abstract

A novel composition for the diagnosis of lung cancer is provided to evaluate the diagnosis, progressed state and convalescence of lung cancer by the specific overexpression in the endotheliocyte derived from lung cancer tissue. A composition for the diagnosis of lung cancer contains an antibody or a polypeptide which is combined specifically to any one protein selected from IGHA-1 (Immunoglobulin Heavy Constant Alpha-1, IPI accession number: IPI00061977.1), IVD (Isovaleryl Coenzyme A Dehydrogenase, IPI accession number: IPI00032297.2), MAPRC-2 (Membrane Associated Progesterone Receptor Component 2,IPI accession number: IPI00005202.1), RPS4X (40S Ribosomal Protein S4, X isoform, IPI accession number: IPI00217030.7), NDUFA5 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5, IPI accession number: IPI00412545.4), RPS8(Ribosomal protein S8, IPI accession number: IPI00216587.6), 7kDa protein (IPI accession number: IPI00333233.1), CSA (Coatomer subunit alpha, IPI accession number: IPI00295857.6), CYFIP-1 (Cytoplasmic FMR1 Interacting protein 1, IPI accession number: IPI00030960.2), SERPINH1 (47kDa heat shock protein precursor, IPI accession number: IPI00019880.1), SF3-S1 (Splicing factor 3 subunit 1, IPI accession number: IPI00017451.1), SILUCTH-5 (Splicing isoform long of ubiquitin carboxyl-terminal hydrolase 5, IPI accession number: IPI00024664.1), EF1-beta (Elongation factor 1-beta, IPI accession number: IPI00178440.2), IGKC (Ig Kappa Chain C Region, IPI accession number: IPI00550315.1), HCV1R HG3 precursor (Similar to Ig heavy chain V-I region HG3 precursor, IPI accession number: IPI00382937.7) and OCIAP (Ovarian carcinoma immunoreactive antigen-like protein, IPI accession number: IPI00555902.1).

Description

Novel biomaker for the diagnosis of lung cancer

The present invention relates to a novel marker for diagnosing lung cancer. More specifically, the present invention Overexpressed in lung cancer tissue-derived endothelial cells The present invention relates to a lung cancer diagnostic composition comprising an antibody specific for a protein and a lung cancer diagnostic composition comprising a primer or probe specific for a nucleic acid encoding the protein.

Lung cancer is the leading cause of cancer deaths worldwide. About one-sixth of all cancer deaths are caused by lung cancer, and survival rates are about 10-15% lower than other types of cancer. Lung cancer is divided into small cell lung cancer and non-small cell lung cancer. Among them, non-small cell lung cancer is the most representative cancer corresponding to about 80% of lung cancers, and is classified into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. In the case of the non-small cell cancer, the 10-year survival rate is very low, less than 10% despite the recent development of cancer therapy. This is because most cancers are diagnosed late. Therefore, there is an urgent need for the development of markers that can increase the survival rate of patients by early diagnosis of lung cancer.

Recently, in order to identify specific genes or proteins that can be used as biomarkers for more advanced diagnosis and treatment, studies using genomics and proteomics analysis comparing normal tissues with diseased tissues themselves have been conducted.

However, this approach creates an excess of molecular information for diagnostic and possible therapeutic targets, making it difficult to sort out useful candidates from large amounts of information. In addition, most biomarkers target complex and inaccessible areas, such as inside tissue. Therefore, new research is needed to find and identify accessible tissue specific targets in order to reduce tissue data complexity and overcome the difficulty of accessing diseased tissue. Accordingly, researches targeting endothelial cells, which are the interface between blood and internal cells in tissues, are being made rather than difficult and complex cells because they are located in tissues.

The literature discloses aminopeptidase-P and annexin A1 as cancer-specific endothelial cell markers (Oh, P. et al., Nature , 429, 2004). Stage or type specific endothelial cell molecules have been identified (Joyce, JA, et al., Cancer Cell , 4, 2003).

However, to date, studies on endothelial specific molecules of lung cancer are insufficient. This is because of the low percentage of cells in lung cancer tissue, which has technical limitations in the molecular profiling of endothelial cells.

Therefore, the present inventors have studied to develop a new diagnostic marker for diagnosing lung cancer. As a result, the proteome of endothelial cells derived from human lung cancer tissues and endothelial cells derived from normal lung tissues was compared and analyzed using proteomic analysis. The present invention was completed by identifying a new biomarker specifically detected only in lung cancer tissue-derived endothelial cells.

Accordingly, an object of the present invention is to provide a novel diagnostic marker for lung cancer.

In order to achieve the object of the present invention as described above, the present invention provides a lung cancer diagnostic composition comprising an antibody specific for a protein overexpressed in lung cancer tissue-derived endothelial cells.

The present invention also provides a composition for diagnosing lung cancer comprising a primer or probe specific for a nucleic acid encoding a protein overexpressed in lung cancer tissue-derived endothelial cells.

Hereinafter, the present invention will be described in detail.

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether lung cancer has developed.

As used herein, the term "lung cancer" refers to a malignant tumor occurring in the lung and preferably, adenocarcinoma among non-small cell lung cancer, more preferably non-small cell lung cancer.

As used herein, the term "cancer diagnostic marker" refers to an organic biomolecule which shows an increase in cancer-causing cells as compared with normal cells as a substance capable of diagnosing cancer cells from normal cells. The organic biomolecules include, but are not limited to, polypeptides, proteins, nucleic acids, lipids, glycolipids, glycoproteins, sugars, and the like. In the present invention, the marker for diagnosing cancer may be a protein or a nucleic acid encoding the same, which specifically expresses excessively in endothelial cells derived from lung cancer tissue.

To identify new lung cancer diagnostic markers, the inventors analyzed proteome of endothelial cells derived from human lung cancer tissue using proteomics technology, and compared them with the proteome of endothelial cells derived from normal tissue. Specific proteins were identified. On the other hand, since endothelial cells are small tissues, it is difficult to separate them from one organ (Garlanda C. and Dejana E, Arteroscler Thromb Vasc Biol , 17, 1997). In one embodiment of the present invention, endothelial cells of human lung adenocarcinoma and adjacent normal tissues were isolated from each of five patients by immunomagnetic separation using CD31 antibody magnetic beads, an endothelial cell specific marker (see Example 1). .

After separating the endothelial cell protein complexes separated by primary SDS-PAGE (see Example <2-1>), gel bands of each lane were digested with in-gel trypsin (see Example <2-2>). The obtained peptide was fractionated by first reverse phase chromatography and analyzed using electrospray ionization and ion trap mass spectrometry (see Example <2-3>). The analytical data obtained above was searched using an IPI human protein database to identify proteins present in lung cancer tissue-derived endothelial cells and normal tissue-derived endothelial cells, and a dataset of lung cancer tissue-derived endothelial cells was identified. By comparison with the dataset of 130 proteins identified only in endothelial cells derived from lung cancer tissues were identified (see Example <2-4>). The 130 proteins identified above are shown in Table 1.

Furthermore, the present inventors performed subtractive proteomics using ProtAn on data extracted from endothelial cells of lung cancer tissues and adjacent normal tissues (see Example <2-5>). As a result, we identified 44 endothelial-specific proteins derived from lung cancer tissue, and among them, except for proteins identified by single-hit peptides, and whether they are located in the correct molecular mass region of the gel and identified per protein. Taking into account the number of peptides obtained, 20 possible candidate proteins that can be reliably identified only in lung cancer tissue derived endothelial cells were obtained (Table 2). Therefore, since all 20 candidate proteins shown in Table 2 can only be detected in endothelial cells derived from lung cancer tissue, it can be used as a marker for diagnosing lung cancer.

The inventors performed Western blot analysis among the 20 candidate proteins shown in Table 2 above, in particular, to confirm whether COPG was actually detected in endothelial cells derived from lung cancer tissue (see Example 3). As a result, it was confirmed that COPG was detected significantly higher in endothelial cells of lung cancer tissue than in normal tissue (FIG. 8).

Therefore, COPG of the present invention exhibits a particularly high level of expression in lung cancer tissue-derived endothelial cells compared to normal tissue-derived endothelial cells, and thus may be used as a diagnostic marker for lung cancer. On the other hand, in the present invention, all of the proteins shown in Table 2 in addition to the COPG can be used as a marker for diagnosing lung cancer. However, it is desirable to use a marker that is not excessive enough to make medical judgment.

Diagnosis of lung cancer using a lung cancer diagnostic marker according to the present invention can be carried out by confirming the presence of a marker protein or nucleic acid of the present invention in a biological sample.

As a method of confirming the presence of the marker protein of the present invention, various assay methods known in the art may be used. Preferably, the presence of the marker protein of the present invention in a biological sample can be confirmed by measuring the formation of the antigen-antibody complex by contacting the antibody specifically binding to the marker protein of the present invention. As used herein, the term “antigen-antibody complex” refers to the combination of a particular protein in a biological sample with an antibody that specifically recognizes it.

Accordingly, the present invention provides COPG (Coatomer protein complex subunit gamma, IPI accession number: IPI00001890.6), IGHA-1 (Immunoglobulin Heavy Constant Alpha-1, IPI accession number: IPI00061977.1), IVD (Isovaleryl Coenzyme A Dehydrogenase, IPI accession number: IPI00032297.2), TIβ / γ (Thymopoietin isoforms beta / gamma, IPI accession number: IPI00030131.2), MAPRC-2 (Membrane Associated Progesterone Receptor Component 2, IPI accession number: IPI00005202.1), RPS4X (40S Ribosomal Protein S4, X isoform, IPI accession number: IPI00217030.7), NDUFA5 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5, IPI accession number: IPI00412545.4), RPS8 (Ribosomal protein S8, IPI accession number: IPI00216587.6) , 7kDa protein (IPI accession number: IPI00333233.1), CSA (Coatomer subunit alpha, IPI accession number: IPI00295857.6), CYFIP-1 (Cytoplasmic FMR1 Interacting protein 1, IPI accession number: IPI00030960.2), SERPINH1 (47kDa heat shock protein precursor, IPI accession number: IPI00019880.1), SF3-S1 (Splicing) factor 3 subunit 1, IPI accession number: IPI00017451.1), SILUCTH-5 (Splicing isoform long of ubiquitin carboxyl-terminal hydrolase 5, IPI accession number: IPI00024664.1), EF1-β (Elongation factor 1-beta, IPI accession number: IPI00178440.2), IGKC (Ig Kappa Chain C Region, IPI accession number: IPI00550315.1), HCV1R HG3 precursor, IPI accession number: IPI00382937.7) and OCIAP (Ovarian Carcinoma immunoreactive antigen-like protein, IPI accession number: IPI00555902.1) Provides a composition for diagnosing lung cancer comprising an antibody that specifically binds to any one of the proteins selected from the group consisting of.

By "antibody" is meant herein a specific protein molecule directed against the antigenic site. Antibodies used in the present invention include monoclonal or polyclonal antibodies, immunologically active fragments (eg, Fab or (Fab) ₂ fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain Fv molecules, Chimeric antibodies and the like.

Since the marker proteins for diagnosing lung cancer of the present invention are known proteins, the antibodies used in the present invention can be prepared by conventional methods well known in the field of immunology using the known proteins as antigens. Proteins used as antigens of antibodies according to the invention can be extracted or synthesized in nature and can be prepared by recombinant methods based on DNA sequences. When using a recombinant technique, a nucleic acid encoding a protein is inserted into an appropriate expression vector, the host cell is cultured so that the target protein is expressed in the transformant transformed with the recombinant expression vector, and then It can be obtained by recovering the protein.

For example, polyclonal antibodies can be produced by the method of injecting a protein antigen into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such antibodies can be prepared using various warm-blooded animals such as horses, cattle, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats.

Monoclonal antibodies are also known as fusion methods (Kohler and Milstein, European J. Immnunol . 6: 511-519 1976), recombinant DNA methods (US Pat. No. 4816567), and phage antibody libraries (Clackson et al., Nature) . , 352, 624-628, 1991; Marks et al., J. Mol. Biol . 222, 58: 1-597, 1991).

The diagnostic composition of the present invention may further include reagents known in the art for use in immunological analysis in addition to the antibody specific for the protein.

Immunological analysis in the above may include any method that can measure the binding of the antigen and the antibody. Such methods are known in the art and include, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme linked immunosorbent assay (ELISA), immunoblotting, and PAR assays. Farr assay), immunoprecipitation, latex aggregation, erythrocyte aggregation, non-turbidity, immunodiffusion, counter-current electrophoresis, single radical immunodiffusion, immunochromatography, protein chips and immunofluorescence.

Reagents used in immunological analysis include labels, solubilizers, and detergents capable of producing a detectable signal. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

The label capable of generating a detectable signal enables qualitatively or quantitatively measuring the formation of an antigen-antibody complex, examples of which include enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and redox molecules. And radioisotopes can be used. Enzymes include β-glucuronidase, β-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glycosidase, hexokinase, malate dehydrogenase, and glucose-6 Phosphate dehydrogenase, invertase and the like can be used. As the fluorescent substance, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, fluorine isothiocyanate and the like can be used. Ligands include biotin derivatives, and luminescent materials include acridinium esters, luciferin, and luciferase. The microparticles include colloidal gold and colored latex, and the redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone and hydroquinone. Radioisotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like. However, any of those that can be used in immunological assays other than those exemplified above may be used.

In addition, as a method for confirming the presence of the marker protein of the present invention, a peptide that specifically binds to the marker protein of the present invention may be used.

Therefore, the present invention provides a composition for diagnosing lung cancer comprising a peptide specifically binding to the proteins shown in Table 2. Preferably, the present invention provides a composition for diagnosing lung cancer comprising a peptide that specifically binds to COPG.

Preferably, the peptide may be composed of 7-35 amino acids derived from the marker protein of the present invention.

On the other hand, the diagnostic composition of the present invention can be provided in a fixed state using a variety of methods known on a suitable carrier or support to increase the speed and convenience of diagnosis ( Antibodies : A Labotory Manual, Harlow & Lane Cold Spring Harbor, 1988). Examples of suitable carriers or supports include agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamides, polyesters, companion rocks, filter papers, ion exchange resins, plastic films, plastic tubes , Glass, polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylons, cups, flat packs and the like. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible form, for example spherical (bead), cylindrical (test tube or inner surface of the well), planar (sheet, test strip).

Preferably, the lung cancer diagnostic composition of the present invention may be provided in the form of a diagnostic kit, microarray, protein chip.

The diagnostic kit may be provided, for example, in the form of a lateral flow assay kit based on immunochromatography to detect a specific protein in a sample. The lateral flow assay kit includes a sample pad applied to the sample, a release pad coated with a detection antibody, a developing membrane (e.g., nitro) in which the sample moves to separate and an antigen-antibody reaction occurs. Cellulose) or strip, and an absorption pad.

The microarray is generally intended to attach an antibody onto a slide glass surface treated with a specific reagent to detect a protein that specifically attaches to the antibody by an antigen-antibody reaction.

In one embodiment of the present invention, Western blot analysis was performed on an endothelial cell derived from lung cancer tissue using an antibody specific for COPG protein (Santa Cruz company) (see Example 3). As a result, it was confirmed that COPG protein is significantly higher in lung cancer tissue-derived endothelial cells than in normal tissue-derived endothelial cells (see FIG. 8).

In addition, the present invention is COPG (Coatomer protein complex subunit gamma), IGHA-1 (Immunoglobulin Heavy Constant Alpha-1), IVD (Isovaleryl Coenzyme A Dehydrogenase), TIβ / γ (Thymopoietin isoforms beta / gamma), MAPRC-2 (Membrane) Associated Progesterone Receptor Component 2), Ribosomal Protein S4 (X isoform), NDUFA5 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5), RPS8 (Ribosomal protein S8), 7kDa protein, Coatomer subunit alpha (CSA), CYFIP-1 (Cytoplasmic FMR1 Interacting protein 1), SERPINH1 (47 kDa heat shock protein precursor), TABCSFBI-2 (Transporter 2, ATP-binding cassette, subfamily B isoform 2), SF3-S1 (Splicing factor 3 subunit 1), SILUCTH-5 ( Splicing isoform long of ubiquitin carboxyl-terminal hydrolase 5, EF1-β (Elongation factor 1-beta), IG KC (Ig Kappa Chain C Region), HCV1R HG3 precursor (Similar to Ig heavy chain VI region HG3 precursor) and OCIAP (Ovarian carcinoma immunoreactive antigen-like protein) It provides a cancer diagnostic composition comprising a specific primer or probe to screen a nucleic acid.

The detection of a specific nucleic acid using a primer may be performed by amplifying the sequence of the gene of interest using an amplification method such as PCR, and then checking whether the gene is amplified by a method known in the art. In addition, the detection of a particular nucleic acid using a probe may be performed by contacting a sample nucleic acid with a probe under suitable conditions and confirming the presence of the hybridizing nucleic acid.

The "primer" refers to a nucleic acid sequence having a short free hydroxyl group, which forms a base pair with a complementary template, and serves as a starting point for template strand copying. Primers of the invention can be chemically synthesized using methods known in the art, such as, for example, phosphoramidite solid support methods.

The "probe" refers to a nucleic acid fragment such as RNA or DNA consisting of several to several hundred bases that can specifically bind to mRNA and is labeled to confirm the presence or absence of a specific mRNA. Probes can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, and the like, and can be labeled with biotin, FITC, rhodamine, DIG, or the like, or radioisotopes.

The probe may also be labeled with a detectable substance, for example, a radiolabel that provides a suitable signal and has sufficient half-life. Labeled probes can be hybridized to nucleic acids on a solid support as known from Sam et al., Molecular Cloning , A Laboratory Mannual, 1989.

As a method for detecting a specific nucleic acid using the probe or primer, for example, but not limited to, polymerase chain reaction (PCR), DNA sequencing, RT-PCR, primer extension method (Nikiforeov et al., Nucl Acids Res 22, 4167-4175, 1994), oligonucleotide extension assays (Nickerson et al., Pro Nat Acad Sci USA, 87, 8923-8927, 1990), allele specific PCR (Rust et al., Nucl) Acids Res , 6, 3623-3629, 1993), RNase mismatch cleavage (Myers et al., Science , 230, 1242-1246, 1985), single strand conformation lymorphism, Orita et al. , Pro Nat Acad Sci USA, 86, 2766-2770, 1989), simultaneous analysis of SSCP and heteroduplex (Lee et al., Mol Cells , 5: 668-672, 1995), modified gradient gel electrophoresis (DGGE, Cariello) et al., Am J Hum Genet , 42, 726-734, 1988), denaturing high performance liquid chromatography, Underhill et al., Genome Res , 7, 996-1005, 1997), hybridization reactions, DNA chips and the like. Examples of such hybridization reactions include Northern hybridization (Maniatis T. et al., Molecular Cloning , Cold Spring Habor Laboratory, NY, 1982), in situ hybridization (Jacquemier et al., Bull Cancer , 90: 31-8 , 2003) and microarrays (Macgregor, Expert Rev Mol Diagn 3: 185-200, 2003).

The lung cancer diagnostic composition of the present invention may further include a reagent generally used in the method for detecting the above-described nucleic acid. For example, metal ion salts such as deoxynulceotide triphosphate (dNTP), a heat resistant polymerase, and magnesium chloride required for a PCR reaction may be included, and include dNTP, a sequenase, etc. required for sequencing. can do.

Preferably, the lung cancer diagnostic composition of the present invention may be provided in the form of a diagnostic kit, a microarray and a DNA chip.

For example, but not limited to, RT-PCR kit including each primer pair specific for the marker gene of the present invention, DNA chip including the substrate to which the cDNA or oligonucleotide of the marker gene of the present invention is attached, and the like. There is this.

As described above, since the lung cancer diagnostic marker of the present invention is specifically overexpressed in lung cancer tissue-derived endothelial cells, it is very useful for diagnosing lung cancer, progression of disease, and prognosis before and after treatment.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Example 1>

Isolation of Endothelial Cells from Human Lung Tissue

<1-1> Isolation of Endothelial Cells from Lung Adenocarcinoma and Adjacent Normal Lung Tissues

Lung adenocarcinoma tissue and adjacent normal lung tissue were obtained from five patients undergoing cancer surgery at Kyungpook National University Hospital. The tissue was cut into fine pieces using a surgical knife and washed with 0.1% BSA / PBS to remove blood and unnecessary tissue. The tissue was then pulverized using a glass grinder (glass grinder, Kontes, USA) to produce single cells, and the tissue residue was filtered by filtration of the pulverized tissue with a cell filter (BD Falcon, USA). The filtered ground tissue was repeatedly washed by centrifugation at 700 rpm, 4 ° C. for 5 minutes, and the cell pellet was suspended in 0.1% BSA / PBS.

Human microvascular endothelial cells were isolated from the lung tissue prepared above using an immunomagnetic separation method using magnetic beads coated with CD31 antibody. CD31-immunomagnetic beads (Dynal, Norway) were added to the single cell suspension and incubated with gentle stirring at 4 ° C. for 40 minutes. After incubation, the culture solution was placed in a magnetic separation device for 1 minute to separate endothelial cells from the composite single cell suspension by very carefully discarding the supernatant from the opposite side of the beads. The pellet was resuspended by repeatedly pipetting into 0.1% BSA / PBS in the same volume as the discarded volume. The isolated endothelial cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% trypto X-100) containing the protease inhibition cocktail, incubated at 4 ° C. for 20 minutes, and then at 4 ° C. Centrifugation at 1200 rpm for 20 minutes. The total protein concentration in the supernatant was measured with a protein assay kit using BSA as standard.

As a result, an average of 4 × 10 ⁵ rosetted endothelial cells were isolated from about 1 to 1.5 g of lung adenocarcinoma tissue and adjacent normal lung tissue, respectively (FIG. 1).

<1-2> Purity Analysis of Isolated Endothelial Cells

The purity of endothelial cells isolated in Example <1-1> was analyzed by Western blot using an antibody against endothelial nitric oxide synthase (eNOS), a known endothelial cell specific protein. At this time, total lung tissue lysate (TLC) and human umbilical vascular endothelial cells were used as a positive control for comparison with isolated endothelial cells, and human lung adenocarcinoma cell line as a negative control. A549 was used. Β-actin was used as a loading control.

As a result, in the human pulmonary vascular endothelial cells obtained in Example <1-1>, eNOS showed a lower expression level than human umbilical vascular endothelial cells (HUVECs), which is an established cell line, but was specifically detected (FIG. 2). ).

<Example 2>

Proteome Analysis

<2-1> One-Dimensional Gel Electrophoresis and Coomassie Dyeing

To analyze endothelial proteome derived from human lung adenocarcinoma tissue and adjacent lung normal tissue, total protein was extracted from five lung adenocarcinoma tissues and adjacent normal tissue derived endothelial cells, respectively. Protein mixtures isolated from the endothelial cells were separated by one-dimensional gel electrophoresis and visualized by Coomassie staining. That is, the endothelial cells obtained in Example 1 were reduced SDS sample buffer (63 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue and 1:20 (v / v) β-meth Captoethanol), boiled for 5 minutes, extracted, and then separated by electrophoresis on 10% or 12% polyacrylamide gel. After electrophoresis, the proteins were visualized by staining with Coomassie Brilliant Blue G-250 staining solution. The gel lanes were then cut into 10-15 consecutive slices of the same size to analyze all proteins that could be detected in the endothelial cells. The SDS-PAGE results are shown in FIG. 3.

<2-2> In-gel trypsin digestion

Tryptic in-gel digestion was performed using the gel obtained in Example <2-1> as a sample. The gel slices were washed with 50% (v / v) acetonitrile dissolved in 0.1 M ammonium bicarbonate and 0.1 M ammonium bicarbonate until the SDS and Coomassie blue dyes were completely removed. The gel slices were then dehydrated in acetonitrile and shrunk and dried in a vacuum centrifuge. Proteins in the shrunken gel were placed in 25 mM ammonium bicarbonate at pH 8.0 and digested overnight with trypsin treatment to a substrate / enzyme ratio of 10: 1 (wt / wt). The enzyme reaction was terminated by treatment with 0.1% formic acid dissolved in water. Peptides derived from the gel pieces were extracted by sonication for 10 minutes and the supernatant containing peptides was transferred to a new tube.

<2-3> uLC-ESI-MS / MS Analysis

Tryptic digested peptides were analyzed in Example <2-2> using a 2D-LC-MS / MS system equipped with reverse phase-liquid chromatography (RP-LC) for proteome analysis. The system includes a Surveyor MS pump, Thermo Electron, San Jose, Calif., USA, Spark auto sampler, Thermo Electron, San Jose, Calif., USA, and nanospray ionization (NSI). It consists of a Finnigan LTQ linear ion trap mass spectrometer with source, Thermo Electron, San Jose, CA, USA. Trypsin digested peptide dissolved in 12 μl of 0.1% formic acid was injected into LC-MS / MS for analysis. First, a digested sample for peptide concentration and desalting is injected into a peptide CapTrap cartirige, and the peptide is separated by hydrophobicity on a reverse phase (RP) column, 5 µm in diameter, 300 mm C18 silica gel. The RP column packed in this house was loaded. The RP column was installed directly in front of the ion transfer tube for electrospray. As the mobile phase, solutions A (H ₂ O) and B (acetonitrile, ACN) were used respectively, and both solutions contained 0.1% (v / v) formic acid. The flow rate was maintained at 200 nL / min. The solvent gradient started with a 5% B solution in 43 minutes, followed by a linear gradient up to 65% B solution, then pulled up to 80% B solution for 7 minutes and then using 100% A solution for 10 minutes. The mass spectrometer was operated in data-dependent mode (m / z 300-1800) where five full MS / MS scans followed by each full MS scan. In this mode, the five most peptide molecular ions are selected from previous scans for collision-induced dissociation (CID) using 35% normalized collision energy. The temperature and electron injection voltage of the heated capillary were 195 ° C. and 2.0 kV, respectively.

<2-4> Biological Information Analysis

The MS / MS data obtained in Example <2-3> was retrieved from the IPI human protein database using the SEQUEST algorithm (Thermo Electron, San Jose, CA) with BioWorks software (version 3.2). This database search allows for various variations of carboxyamidomethylation on cysteine residues (57Da) and oxidation on methionine residues (16Da) and peptide mass error 2Da and fragment mass error 1Da. The database search is limited to peptides produced by trypsin cleavage. The SEQUEST results were filtered by Xcorr for the charge step. Xcorr values were used to match 1.9 for single ionized ions, 2.2 for double ionized ions and 3.75 for triple ionized ions. Delta Cn ≧ 0.1 and Rsp ≦ 4 and Probability score ≧ 1.0E-3. False positive identification was removed by filtering on the peptides identified under the above conditions. As a result, about 550 to 1052 proteins (average 790 proteins) were identified for each sample.

In order to select more reliable proteome, second order filtering was performed by applying stringency higher than peptide ≧ 2 for the primary filtered dataset above. That is, a protein in which two or more peptides were identified in the first filtered dataset was regarded as positive identification. As a result, about 400-736 proteins of protein were identified for each sample (Fig. 4).

As described above, normal tissue-derived endothelial cells and cancer tissue-derived endothelial cells were filtered with peptide≥2, and then the common proteins detected in three or more samples (60%) of five protein identification datasets were collected. Reduced. As a result, 449 proteins (45%) and 498 proteins (40%) were extracted from 996 proteins in the merged normal dataset and 1233 proteins in the merged cancer dataset, respectively.

The dataset of lung cancer tissue derived endothelial cells reduced by this method was compared with the dataset of normal tissue derived endothelial cells. As a result, 368 proteins (74%) out of 498 proteins of lung cancer tissue-derived endothelial cells were expressed in normal, and 130 proteins were found to exist only in endothelial cells derived from lung cancer tissue (FIG. 5).

Table 1 lists some of the lung cancer tissue endothelial specific proteins identified in the present invention, along with their subcellular location and function. 130 lung cancer tissue-derived endothelial specific proteins and 81 normal tissue-derived endothelial protein proteins were classified according to their intracellular location and physiological processes (FIG. 6). As a result, in the case of the intracellular location, the cytoplasmic reticulum and ribosomal-derived protein were 6.4 times and 5.4 times more in the cancer-specific protein than in the normal cell protein, respectively. For physiological processes, metabolic related proteins in cancer cell-specific proteins were detected 1.7 times more than normal cell proteins. In particular, among them, a significant number of protein metabolism related ribosomal proteins were found.

Table 1. Proteins specifically identified in lung cancer tissue-derived endothelial cells

<2-5> Subtraction Analysis

In order to identify proteins expressing specifically in endothelial cells derived from lung cancer tissues, subtractive proteomics was performed using ProtAn on five individual data extracted from endothelial cells of lung cancer tissues and adjacent normal tissues. Cancer tissue-derived endothelial protein showed an average of 59% homology to normal tissue-derived endothelial protein (FIG. 7). Integral assays were performed on individually extracted cancer-specific proteins to explore proteins commonly expressed in other types of cancer datasets. Expected protein expressed in all five samples but was not found. Instead, 85 proteins detected in three or more samples of the five sample dataset could be collected. Of the 85 proteins, 44 were not detected in normal tissue-derived endothelial proteins. Of the 44 proteins, nine were shown to contain proteins identified by single-hit peptides. Next, the protein was located in the correct molecular mass region of the gel and additionally considered the number of peptides identified per protein. As a result, 20 possible candidate proteins that can be reliably identified only in cancer tissue derived endothelial cells were obtained (Table 2).

In order to find a more robust candidate protein, the three classes of upper, middle, and lower layers were determined according to the degree of satisfying the number of proteins identified by peptide ≥ 2 and the number of identified peptides derived from the exact molecular mass region of the gel. Divided.

Table 2. Biomarkers of Endothelial Cells Derived from Lung Cancer Tissues

<Example 3>

Confirmation by Western Blot Analysis of Candidate Proteins

<3-1> Western blot analysis of COPG

Among the 20 candidate proteins shown in Table 2, it was confirmed by Western blot analysis whether the coater protein gamma (COPG) was specifically detected only in endothelial cells derived from lung cancer tissue. The COPG belongs to a subgroup (Table 2), but had an SEQUEST score of 27.6 on average and was specifically identified in four cancer tissue derived endothelial cells. Therefore, COPG is detected in endothelial cell proteins derived from samples 2, 4 and 5 identified by MS / MS analysis, and in addition to endothelial cell proteins (extra-case 1, 2 and 3) derived from three lung cancer tissues. Whether or not was confirmed using an antibody specific for COPG. At this time, endothelial protein of normal tissue adjacent to the lung cancer tissue was used as a control, and β-actin was used to monitor whether the same amount of sample was injected into the gel. Western blot analysis was performed by loading each sample protein on an SDS gel, followed by electrophoresis, and then transferring the protein on the gel to a nitrocellulose membrane, followed by treatment with a COPG antibody (Santa Cruz biotechnology, CA, USA).

As a result, it was found that COPG was significantly higher in endothelial cells of lung cancer tissue than in normal tissue (FIG. 8).

<3-2> Western blot analysis of Prdx IV

It was confirmed by Western blot analysis whether peroxiredoxin IV (Prdx IV) is specifically expressed only in endothelial cells of lung cancer tissue. The Prdx IV belonged to a subgroup (Table 2), but had a slightly lower SEQUEST score (mean 16.8) and was cancer-specific in three lung cancer tissue derived endothelial samples. Western blot analysis of the Prdx IV was performed in the same manner as in Example <3-1>, but using a specific antibody (Santa Cruz biotechnology, CA, USA) to Prdx IV.

As a result, Prdx IV was found to be expressed higher in lung cancer tissue-derived endothelial cells than normal tissue-derived endothelial cells (FIG. 9).

1 shows human microvascular endothelial cells isolated from lung cancer patients using an immunomagnetic separation method.

A: (a) lung cancer tissue, (b) adjacent normal tissue

B: Single Cell Isolated from Lung Tissue

C: CD31-immunomagnetic beads bound to endothelial cells

Figure 2 shows the endothelial cells isolated in the present invention analyzed by Western blot using endothelial specific endothelial nitric oxide synthase (eNOS) antibody.

TCL: Total Lung Tissue Lysate

HLVEC: Human lung capillary endothelial cells isolated from the present invention

HUVEC: human umbilical cord endothelial cells

A549: human lung cancer cell line

Figure 3 shows the results of one-dimensional gel electrophoresis of proteins extracted from endothelial cells derived from five human lung cancer tissues and adjacent normal tissues.

SM: Size Marker

N: normal tissue-derived endothelial protein

C: endothelial protein derived from lung cancer tissue

4 is a result of analyzing the proteome of human lung cancer tissue and normal tissue-derived endothelial cells.

5 is a result of comparing the proteome of human lung cancer tissue-derived endothelial cells with that of normal tissue-derived endothelial cells.

N: normal tissue-derived endothelial cells

C: endothelial cells derived from lung cancer tissue

N1-N5: normal tissue derived endothelial cells 1 to 5

C1-C5: endothelial cells derived from lung cancer tissue 1 to 5

6 shows proteins identified from normal tissue-derived endothelial cells (A and C) and lung cancer tissue-derived endothelial cells (B and D) according to the present invention at intracellular locations (A and B) and biological processes (C and D). It is shown separately according to.

7 shows the results of individually comparing proteins identified from endothelial cells derived from five lung cancer tissues and endothelial cells derived from normal tissues, respectively, according to the method of the present invention.

8 is a result of confirming the expression of COPG (Coatomer protein complex subunit gamma) in lung cancer tissue-derived endothelial cells and normal tissue-derived endothelial cells by Western blot analysis.

9 shows the results of Western blot analysis of the expression of Prdx IV (peroxiredoxin IV) in lung cancer tissue-derived endothelial cells and normal tissue-derived endothelial cells.

Claims (3)

Immunoglobulin Heavy Constant Alpha-1, IGHA-1 (IPI accession number: IPI00061977.1), IVD (Isovaleryl Coenzyme A Dehydrogenase, IPI accession number: IPI00032297.2), MAPRC-2 (Membrane Associated Progesterone Receptor Component 2, IPI accession number : IPI00005202.1), RPS4X (40S Ribosomal Protein S4, X isoform, IPI accession number: IPI00217030.7), NDUFA5 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5, IPI accession number: IPI00412545.4), RPS8 (Ribosomal protein S8, IPI accession number: IPI00216587.6), 7kDa protein (IPI accession number: IPI00333233.1), CSA (Coatomer subunit alpha, IPI accession number: IPI00295857.6), CYFIP-1 (Cytoplasmic FMR1 Interacting protein 1, IPI accession number: IPI00030960.2), SERPINH1 (47kDa heat shock protein precursor, IPI accession number: IPI00019880.1), SF3-S1 (Splicing factor 3 subunit 1, IPI accession number: IPI00017451.1), SILUCTH-5 (Splicing isoform long of ubiquitin carboxyl-terminal hydrolase 5, IPI accession number: IPI00024664.1), EF1-β (Elongati on factor 1-beta, IPI accession number: IPI00178440.2), IGKC (Ig Kappa Chain C Region, IPI accession number: IPI00550315.1), HCV1R HG3 precursor (Similar to Ig heavy chain VI region HG3 precursor, IPI accession number: IPI00382937.7) and OCIAP (Ovarian carcinoma immunoreactive antigen-like protein, IPI accession number: IPI00555902.1) lung cancer diagnostic composition comprising an antibody that specifically binds to any one selected from the group consisting of. Immunoglobulin Heavy Constant Alpha-1, IGHA-1 (IPI accession number: IPI00061977.1), IVD (Isovaleryl Coenzyme A Dehydrogenase, IPI accession number: IPI00032297.2), MAPRC-2 (Membrane Associated Progesterone Receptor Component 2, IPI accession number : IPI00005202.1), RPS4X (40S Ribosomal Protein S4, X isoform, IPI accession number: IPI00217030.7), NDUFA5 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5, IPI accession number: IPI00412545.4), RPS8 (Ribosomal protein S8, IPI accession number: IPI00216587.6), 7kDa protein (IPI accession number: IPI00333233.1), CSA (Coatomer subunit alpha, IPI accession number: IPI00295857.6), CYFIP-1 (Cytoplasmic FMR1 Interacting protein 1, IPI accession number: IPI00030960.2), SERPINH1 (47kDa heat shock protein precursor, IPI accession number: IPI00019880.1), SF3-S1 (Splicing factor 3 subunit 1, IPI accession number: IPI00017451.1), SILUCTH-5 (Splicing isoform long of ubiquitin carboxyl-terminal hydrolase 5, IPI accession number: IPI00024664.1), EF1-β (Elongati on factor 1-beta, IPI accession number: IPI00178440.2), IGKC (Ig Kappa Chain C Region, IPI accession number: IPI00550315.1), HCV1R HG3 precursor, IPI accession number : IPI00382937.7) and OCIAP (Ovarian carcinoma immunoreactive antigen-like protein, IPI accession number: IPI00555902.1) lung cancer diagnostic composition comprising a polypeptide specifically binding to any one selected from the group consisting of. Immunoglobulin Heavy Constant Alpha-1, IGHA-1 (IPI accession number: IPI00061977.1), IVD (Isovaleryl Coenzyme A Dehydrogenase, IPI accession number: IPI00032297.2), MAPRC-2 (Membrane Associated Progesterone Receptor Component 2, IPI accession number : IPI00005202.1), RPS4X (40S Ribosomal Protein S4, X isoform, IPI accession number: IPI00217030.7), NDUFA5 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5, IPI accession number: IPI00412545.4), RPS8 (Ribosomal protein S8, IPI accession number: IPI00216587.6), 7kDa protein (IPI accession number: IPI00333233.1), CSA (Coatomer subunit alpha, IPI accession number: IPI00295857.6), CYFIP-1 (Cytoplasmic FMR1 Interacting protein 1, IPI accession number: IPI00030960.2), SERPINH1 (47kDa heat shock protein precursor, IPI accession number: IPI00019880.1), SF3-S1 (Splicing factor 3 subunit 1, IPI accession number: IPI00017451.1), SILUCTH-5 (Splicing isoform long of ubiquitin carboxyl-terminal hydrolase 5, IPI accession number: IPI00024664.1), EF1-β (Elongat ion factor 1-beta, IPI accession number: IPI00178440.2), IGKC (Ig Kappa Chain C Region, IPI accession number: IPI00550315.1), HCV1R HG3 precursor (Similar to Ig heavy chain VI region HG3 precursor, IPI accession number: IPI00382937.7) and OCIAP (Ovarian carcinoma immunoreactive antigen-like protein, IPI accession number: IPI00555902.1) Lung cancer comprising a primer or probe that specifically binds to a gene encoding a protein selected from the group consisting of Diagnostic composition.

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