KR100925147B1 - Markers for the diagnosis of lung cancer - Google Patents

Abstract

PURPOSE: A composition for detecting a lung cancer marker containing an agent which measures gene expression is provided to diagnose lung cancer and enable qualitative analysis. CONSTITUTION: A method for detecting a lung cancer marker comprises: a step of calculating mass of peptide which is cut in a biological sample; and a step of comparing mass spectrum of peptide with protein sequence data base; The biological sample is tissue, cell, whole blood, blood serum, plasma, sputum or urine.

Description

Markers for the diagnosis of lung cancer

The present invention relates to a composition for detecting a lung cancer marker comprising an agent for measuring expression of a gene overexpressed in lung cancer, which is a tumor marker, and a method for diagnosing lung cancer using the same.

Cancer is not easy to detect early and its treatment is difficult, so the mortality rate from cancer is the number one, and the incidence rate is steadily increasing. Lung cancer is the second most common cancer.

Serum amyloid A (SAA) is known as a precursor to the inflammatory situation of reactive amyloidosis and is the major acute phase protein with increased serum concentrations in various body conditions. The SAA multigene family contains four similar genes, of which SAA1 and SAA2 are the main proteins in acute phase response, with SAA3 being a pseudo-gene and SAA4 being persistent. It is known to be expressed.

SAA1 and SAA2 are on the same chromosome and have a very high genetic homology, resulting in only 9 unique amino acids out of a total of 122 amino acids. But these two proteins are different gene genes expressed by different parts of the DNA. Many studies on SAA have been conducted to date but are mostly for SAA1, and the usefulness of SAA2 as a biomarker has not been studied yet.

In addition to SAA2, there is a need for the discovery of biomarkers that can be used for significant diagnosis, prognostic observation and treatment of lung cancer.

Therefore, we want to discover a significant biomarker that can easily and effectively diagnose lung cancer.

In the present invention, proteins that are specifically overexpressed in a biological sample obtained from a lung cancer patient compared to a normal person were screened, thereby securing the genes described in Table 2 as lung cancer markers.

That is, the present invention provides serum amyloid A2, complement C4-A precursor, serum amyloid A protein precursor, and complement component C8 gamma chain precursor. gamma chain precursor), 187 kDa protein, hypothetical protein DKFZp686C15213, hypothetical protein DKFZp686N02209 (Hypothetical protein DKFZp686N02209), apolipoprotein A-II precursor, Apolipoprotein A-II precursor Isoform 1 of Alpha-1-antichymotrypsin precursor, Platelet basic protein precursor, Serotransferrin precursor, Leucine-Rich alpha-2-glycoprotein precursor (Leucine-rich alpha-2-glycoprotein precursor), Vitamin D-binding protein precursor, SNC66 protein, IGL @ protein (Ig lamda chain protein), Isoform 1 of C-reactive protein precursor, alpha-2-glycoprotein 1, zinc, Clusterin precursors), Apolipoprotein E precursors, and Isoform 2 of Apolipoprotein-L1 precursors are provided as biomarkers of lung cancer.

The term “lung cancer marker” or “lung cancer biomarker” refers to an organic biomolecular substance capable of diagnosing lung cancer by showing an increase or decrease in cells, tissues, and the like with lung cancer as compared to normal cells. Lung cancer can be diagnosed by confirming the expression of the described lung cancer biomarkers at the mRNA level or the protein level. At this time, the number of genes used for analysis is not limited, and one or more genes can be appropriately selected according to the purpose.

In one aspect, the present invention relates to a composition for detecting a lung cancer diagnostic marker comprising an agent for measuring the mRNA level of 1 to 20 genes selected from the group consisting of the genes listed in Table 2.

The agent for measuring mRNA level of the gene is preferably a primer pair or probe.

Specifically, the primer is a nucleic acid sequence having a short free 3 'hydroxyl group, which forms a complementary template and base pair and serves as a starting point for template strand copying. Means.

The primers of the present invention may, if necessary, comprise a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of the label include enzymes such as horseradish peroxidase, alkaline phosphatase, and the like, radioisotopes such as 33P, chemical groups such as biotin, and fluorescent molecules.

Probe means a nucleic acid fragment such as RNA or DNA that can achieve specific binding with mRNA and can be labeled to confirm the presence or absence of a specific mRNA. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like.

A person skilled in the art can specifically recognize a specific region of the gene or a primer pair that specifically amplifies a specific region of the gene in consideration of variables such as the degree of hybridization between sequences in the target gene from nucleic acid sequences of known genes. Probes can be designed.

In another aspect, the present invention relates to a composition for detecting lung cancer markers comprising an agent for measuring the protein level of 1 to 20 genes selected from the group consisting of the genes listed in Table 2.

Agents for measuring protein levels of genes are preferably antibodies, which include both polyclonal antibodies, monoclonal antibodies and recombinant antibodies that specifically bind to the proteins of the genes listed in Table 2. In addition, the antibodies of the present invention are functional fragments of the antibody molecule in full form with two full length light chains and two full length heavy chains, eg, Fab, F (ab '), F (ab') 2 And Fv.

The composition for detecting a lung cancer diagnostic marker of the present invention may further include other components or devices widely used in the art, and may be applied and manufactured as a kit for detecting a lung cancer diagnostic marker.

Specific examples include primer pairs specific for the gene and, in addition, test tubes or other suitable containers, reaction buffers, enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, DNAse inhibitors, RNAse inhibitors, It can be produced as a kit for RT-PCR, including DEPC-water, sterile water and the like. As another example, the kit may be manufactured as a kit for a DNA chip containing essential elements necessary for carrying out the DNA chip.

As another example, the antibody comprises a specific antibody to a protein of the gene and additionally a reagent capable of detecting the bound antibody, such as labeled secondary antibodies, chromophores, enzymes and substrates thereof. It can be produced as a kit for ELISA. As another example, the kit may be manufactured as a kit for a protein chip containing essential elements necessary for carrying out the protein chip.

In addition to the kits exemplified above, it includes various types of diagnostic kits that are manufactured as rapid diagnostic kits capable of detecting the gene.

In another aspect, the present invention relates to a method for detecting lung cancer markers by measuring the expression level of 1 to 20 genes selected from the group consisting of the genes listed in Table 2 of a biological sample. Specifically, the expression level of the gene can be detected at the mRNA level or the protein level.

In a specific embodiment, the present invention relates to a method for detecting a lung cancer diagnostic marker, comprising measuring mRNA levels of the gene from a biological sample of a suspected lung cancer patient using primer pairs or probes specific for the genes listed in Table 2. It is about.

Specific analytical methods for measuring mRNA levels by the above methods include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, northern blotting, DNA chip, and the like.

In another specific embodiment, the invention comprises measuring protein levels by a method of forming an antigen-antibody complex by contacting a biological sample of a suspected lung cancer patient with an antibody specific for the proteins of the genes listed in Table 2, A method for detecting lung cancer diagnostic markers.

Specific analytical methods for measuring protein levels by the above method, Western blot, ELISA, radioimmunoassay, radioimmunoproliferation method, oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunity staining, immunoprecipitation assay, complement fixation Assays, FACS, protein chips, and the like, but are not limited to such. ELISA is a direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support, followed by reaction with another antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support. Various ELISA methods, such as indirect sandwich ELISA using the labeled secondary antibody which recognizes this antibody, are included.

As another specific embodiment, for the protein selected from the group consisting of 1 to 20, the step of obtaining the mass of the cleaved peptide in the biological sample using a mass spectrometer; And identifying a peptide produced by comparing the mass spectrum of the peptide with a protein sequence database. Specifically, the mass spectrometer employs multiple reaction monitoring (MRM) or selective reaction monitoring (SRM) methods to produce precursor and transition ions of the resulting protein-derived peptide. Identify the peak on the mass spectrometer of the ion. In addition, software such as SEQUEST, Mascot, Sonar, X! Tandem, Phenyx, PeptideProphet, ProteinProphet, DTASelect, and OMSSA can be used.

The method for detecting and measuring lung cancer biomarkers provided by the present invention is not limited to the above-mentioned methods, and may be applied by a combination of various methods and modified methods known in the art. In this way, SISCAPA (stable isotope standards and capture by anti-peptide antibodies) can be exemplified.

The biological sample used for the test is not particularly limited and may include tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, samples such as urine, and the like.

The method may also include comparing the gene expression level in the biological sample of the suspected lung cancer patient in the sample by using the sample that does not have lung cancer as a control. This can be compared with absolute or relative differences in the amount of expression between the control and the biological sample.

Through the above-described detection methods, it is possible to quantitatively analyze mRNA or protein levels of lung cancer marker genes, thereby diagnosing lung cancer of suspected lung cancer patients.

The expression of the genes listed in Table 2 specifically overexpressed in lung cancer can be measured at the mRNA or protein level to effectively diagnose lung cancer.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Materials and Methods

1-1 Serum Samples

Serum samples were taken from five smoking-induced adenocarcinoma patients and five nonsmokers with no cancer identified. These serums were isolated immediately after blood collection and stored in a -70 ° C freezer until the experiment.

1-2 SDS-PAGE and Coomassie Blue Staining

Serum from each group was pooled in equal amounts. The amount of protein contained in the serum was measured by Bradford assay (electrophoresis) to electrophores the same amount of protein. Electrophoresis was performed at 110V for 2 hours on a 15% acrylamide gel.

After the electrophoresis, acrylamide gel was washed with distilled water, and then reacted for two hours while shaking at room temperature with Coomassie G250 Stain (BioRad). The gel was then dyed overnight by decolorizing in distilled water and washing with distilled water.

1-3 in-gel digestion

The protein band of the region to be analyzed identified in corgi staining was cut and decolorized in a destaining solution in which 1: 1 was mixed with 75 mM ammonium bicarbonate and 40% ethanol. 5 mM DTT / 25 mM ammonium bicarbonate was sufficiently added to the gel in which the color of Coomassie Brilliant blue disappeared and reduced at 60 ° C. for 30 minutes, and then all the solutions were removed. After washing once with 25 mM ammonium bicarbonate solution, it was alkylated by reacting with 55 mM iodoacetamide / 25 mM ammonium bicarbonate solution at RT for 30 minutes. The gel pieces were then dehydrated using 100% ACN and dried. The dried gel flakes are loaded with 25 mM ammonium bicarbonate, containing 20 mg / mL modified sequencing grade trypsin (Roche Applied Science), which is sufficiently protein-free and allowed to react overnight at 37 ° C. The next day, 0.1% formic acid is added to elute the protein.

1-4 LC-MS / MS Analysis

LC-MS / MS analysis was performed using Thermo Finnigan's ProteomeX workstation LTQ linear IT MS (Thermo Electron, San Jose, CA, USA). 12 ul of each sample was used and the injected sample was suspended in 5 mm 10 mm RP PicoFrit column packed with 5 mm, 300 A pore size C18, followed by H ₂ O and ACN. It was confirmed by releasing as a gradient solution of.

1-5 Data Analysis

MS / MS samples were analyzed using the SEQUEST algorithm (ThermoFinnigan, San Jose, CA; version v.27, rev. 11) and sequencing was performed based on the ipiHUMAN3.29 database (68161 entries). Scaffold (version Scaffold-01_07_00, Proteome Software Inc., Portland, OR) was used to reconfirm the results of MS / MS. According to the Peptide Prophet algorithm (Keller, A et al Anal. Chem. 2002; 74 (20): 5383-92), those with a probability of more than 90.0% were identified and the Protein Prophet algorithm (Nesvizhskii, AI Anal Chem) According to 2003 Sep 1; 75 (17): 4646-58), the protein was identified based on having a probability of 95.0% or more and two or more peptides.

1-6 Western Blot

100 ug of crude serum protein from each normal subject, lung cancer, other cancer patients, and systemic inflammatory disease was isolated by 15% SDS-PAGE. After electrophoresis, the protein was transferred to PVDF membrane (Millipore, U.S.A.). The membrane was reacted overnight at 4 ° C. with anti-serum amyloid antibody (R & D Systems, Inc. 1: 1000 dilution). After the reaction with an anti-rabbit IgG antibody (Santa Cruz, 1: 3000) for 1 hour at room temperature, the presence of the protein was confirmed by the ECL plus Western blot analysis system (Amersham Biosciences, UK).

Example 2: Protein Separation by SDS-PAGE

100 ug of protein was loaded into each lane after protein quantification so that the same amount of protein was used in the lungs of lung cancer patients and normal subjects. The result of confirming the difference between the two samples after electrophoresis through Coomassie blue staining is a photograph of FIG. As can be seen from the photo, we can see the difference between the two lanes near 45kDa, 15kDa, and 10kDa. The three bands with the difference are expected to have a protein specific to lung cancer, and the protein in the region was analyzed by LC-MS / MS.

Example 3: LC-MS / MS Analysis

Proteins were purified and separated through in-gel digestion process, and the gel was analyzed by LC-MS / MS. The analysis results thus obtained are shown in Table 1. In total, the number of proteins found was about 20 to 30, and about 13 to 20 were common in the same and normal sample bands.

Proteins whose ratio (Fold value) is 1.5 or more when the peptide hits of these found proteins are compared are shown in Table 2. Among these proteins, SAA2 was identified only in lung cancer sample lane band 2, and the peptide hit number was 10 in lung cancer sample lane band 2, compared with no number in normal sample lanes.

Number of proteins identified Peptide identified Peptides in common Compare 1 LC1 21 17 N1 22 Compare 2 LC2 18 13 N2 23 Compare 3 LC3 28 19 N3 26

Example 4: Identification of SAA2

The SAA2 shown by LC-MS / MS analysis was verified through the scaffold (Table 3). Of the 10 peptide fragments shown, there were 3 fragments containing SAA1 and other specific peptides (Underlined Amino Acids-Table 4) and 5 of 9 amino acids (Amino Acids written in Red-Table 4) known to be different. Contained amino acids.

Example 5: Small Group Verification of SAA

Currently available commercially available SAA antibody has no homology with the two proteins due to the high homology shown in FIG. Therefore, it was confirmed using anti-human serum amyloid A antibody. The experiment was conducted in the sera of 10 normal and 50 lung adenocarcinoma patients. As a result, no expression of SAA was observed in 10 normal subjects, and expression was confirmed in 28 patient samples in 50 lung cancer patients.

Example 6: Lung Cancer Specific Expression of SAA Compared to Other Types of Cancer

In order to confirm whether the expression of SAA is specifically expressed in lung cancer patients, the expression of SAA was confirmed in the serum of gastric cancer, rectal cancer, breast cancer, cirrhosis and liver cancer patients. Each sample was pooled with five stage 1, 2, or 3,4 patient sera, which were loaded into each Lane A, B. As a result, the expression of SAA was confirmed only in the serum of lung cancer patients, and the expression of SAA was not observed in the blood of other cancer patients of the same amount.

Example 7: Lung Cancer Specific Expression of SAA Compared to Systemic Inflammatory Disease

SAA is known as an acute phase protein, and therefore its expression is known to be increased by inflammatory responses in the body. Therefore, we compared the expression of SAA in atherosclerosis and diabetic patients with lung cancer patients. As a result, the expression of high SAA was found only in the blood of lung cancer patients, but not in arteriosclerosis patients and diabetic patients.

Since lung cancer biomarkers disclosed in the present invention can be easily and efficiently diagnosed lung cancer, it is expected to be widely used in various medical fields.

FIG. 1 shows SDS-PAGE of each sample to find a protein having a difference in expression between a normal person and a lung adenocarcinoma. As a result, three bands of 45, 15, and 10 kDa were identified at different intensities in the normal and lung cancer patients.

2 is a result of confirming the expression of SAA in the serum of lung adenocarcinoma by Western blot analysis, the band intensity is individual differences according to each patient, but not found in normal people, was expressed high in the blood of lung cancer patients.

Figure 3 shows the results of Western blot analysis of the degree of SAA expression in cancer patient samples, lane A shows stages 1 and 2, lane B shows stages 3 and 4 (LiCi; Liver Cirrhosis, HCC; hepatocellular (hepatocellular) carcinoma)).

Figure 4 shows the Western blot results after loading the original serum proteins of normal, lung cancer patients, arteriosclerosis patients, diabetic patients (DM; diabetes with diabetes mellitus (lane A), diabetes with diabetes mellitus (lane B) )).

Claims (7)

delete delete delete delete For the serum amyloid A2 protein shown in SEQ ID NO: 2, determining the mass of the cleaved peptide in the biological sample using a mass spectrometer; And identifying the generated peptide by comparing the mass spectrum of the peptide with a protein sequence database. The method of claim 5, wherein the biological sample is tissue, cells, whole blood, serum, plasma, saliva, sputum or urine. The method according to claim 5, wherein the mass spectrometer is subjected to multiple reaction monitoring (MRM) or selective reaction monitoring (SRM) methods, using the peaks on the mass spectrometer of the protons and transition ions of the resulting serum amyloid A2 protein-derived peptides. Quantitatively detecting the amyloid A2 protein.

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