JP2008283945A - Marker for detecting hepatic carcinoma - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find a gene and a protein that can be a marker for detecting hepatic carcinoma, and to provide a method for detecting the hepatic carcinoma by measuring the gene and/or the protein to contribute the diagnosis of the hepatic carcinoma. <P>SOLUTION: The method for detecting the hepatic carcinoma includes detecting the TM4SF4 gene and/or the protein encoded by the gene in a test sample, especially a blood sample. A reagent kit is used for the method for detecting the hepatic carcinoma, and the method and kit for diagnosing the hepatic carcinoma using the method for detecting the hepatic carcinoma and the reagent kit are also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a marker for detecting liver cancer, particularly a marker for detecting liver cancer comprising a gene that is highly expressed in hepatocellular carcinoma, and a marker for detecting liver cancer comprising a protein encoded by the gene. More specifically, the present invention relates to a method for detecting or diagnosing liver cancer, wherein the marker is measured on a blood sample. The present invention also relates to a reagent for detecting liver cancer and a reagent kit.

  Hepatocellular carcinoma accounts for more than 90% of primary liver cancer, and in Japan, it is a malignant tumor with a poor prognosis, followed by lung cancer, stomach cancer, and colon cancer. In addition, the number of patients with hepatocellular carcinoma is expected to increase over the next 20 years.

  It is said that hepatocellular carcinoma often develops on the basis of chronic hepatitis or cirrhosis against hepatitis B and C. Therefore, these hepatitis patients are recognized as high-risk groups and are closely monitored. For follow-up of hepatitis patients, imaging tests such as abdominal ultrasonography, CT (Computed Tomography), MRI (Magnetic Resonance Imaging), α-fetoprotein (AFP), AFP L3 fraction, PIVKA- It is recommended to periodically measure markers such as II (Protein Induced by Vitamin-K Absence-II). However, these markers do not always become positive (indicates a high value) with the development of liver cancer, and may increase with cirrhosis or be affected by the drug being administered. Therefore, it can be said that the development of a new marker for detecting liver cancer is an urgent task.

  Transmembrane 4 Superfamily 4 (transmembrane 4 superfamily 4, hereinafter abbreviated as TM4SF4) gene has been confirmed to be expressed in the liver, intestine and pancreas (Non-patent Document 1). In a rat hepatectomy model, high expression of TM4SF4 has been observed after liver resection (Non-patent Document 2). It has also been reported that TM4SF4 expression is observed in oval cells that are considered to be liver stem cells in rat experimental hepatitis models (Non-patent Document 3).

  The protein encoded by the TM4SF4 gene does not have a signal transduction domain and is a membrane protein having four transmembrane domains, but its function in the living body has not been fully elucidated. The TM4SF4 gene and the membrane protein encoded by the gene have very high sequence homology in human, rat and mouse.

Wiseman Institute of Science Public Database (http://genome-www.stanford.edu/cgi-bin/genecards/carddisp.pl?gene=TM4SF4). Liu Z. et al., “BIOCHIMICA ET BIOPHYSICA ACTA”, March 19, 2001, No. 1518, No. 1-2, p. 183-189. Qiu J. et al., “Journal of Hepatology”, February 2007, 46, No. 2, p. 266-275.

  An object of the present invention is to find a gene and protein that can serve as a marker for detection of liver cancer, and to provide a method for detecting liver cancer characterized by measuring the gene and / or protein as a marker, thereby contributing to the diagnosis of liver cancer There is.

  The present inventors focused on a gene group that changes in the carcinogenesis process in a mouse liver carcinogenesis model, screened the gene group with a microarray, and as a result, found that the expression of TM4SF4 gene is increased in cancer tissues. . In addition, the present inventors have demonstrated that TM4SF4 gene is expressed in cancer cells in a mouse liver carcinogenesis model.

  Furthermore, the present inventors have found that the TM4SF4 gene is highly expressed in liver cancer tissues of human liver cancer patients as compared with normal liver tissues.

  From these results, the present inventors have found that the TM4SF4 gene and a polynucleotide encoded by the gene can be used as a marker for detecting liver cancer.

  Furthermore, the present inventors have demonstrated that TM4SF4 mRNA is detected in peripheral blood mononuclear cells of human hepatocellular carcinoma patients, but is not detected in healthy human peripheral blood mononuclear cells.

  From the above results, it was found that liver cancer can be detected and diagnosed by measuring TM4SF4 gene expression in blood samples, and the present invention has been achieved.

  That is, the present invention relates to a method for detecting liver cancer, comprising measuring the expression of TM4SF4 gene in a blood sample.

  The present invention also relates to a method for detecting liver cancer, comprising measuring TM4SF4 gene expression in a blood sample, wherein the primer or probe specifically hybridizes the measurement of TM4SF4 gene expression to mRNA or cDNA of the gene. It is related with the method including performing using.

Furthermore, the present invention is a method for detecting liver cancer, comprising measuring TM4SF4 gene expression in a blood sample, and measuring TM4SF4 gene expression using at least one of the following primer sets: Regarding methods involving:
(1) A primer set composed of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 3 in the sequence listing and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 4.
(2) A primer set composed of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 5 in the sequence listing and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 6; and (3) a sequence set forth in the sequence listing. A primer set composed of an oligonucleotide represented by the base sequence described in No. 7 and an oligonucleotide represented by the base sequence described in SEQ ID No. 8.

Furthermore, the present invention relates to a method for detecting liver cancer, comprising measuring TM4SF4 gene expression in a blood sample, wherein the measurement of TM4SF4 gene expression comprises the following steps:
(I) A primer set comprising a TM4SF4 gene cDNA fragment polynucleotide comprising an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing and an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 4 A step of forming a primary amplification product by amplification by
(Ii) A primer set comprising a fragment polynucleotide of the primary amplification product consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 5 in the sequence listing and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 6 A step of forming a secondary amplification product by amplification by (iii), and a step of detecting the secondary amplification product.

  The present invention also relates to a method for detecting liver cancer, comprising measuring TM4SF4 gene expression in a blood sample, wherein TM4SF4 gene expression is measured by measuring a protein encoded by the gene. Relates to the method of inclusion.

  Furthermore, the present invention is a method for detecting liver cancer, comprising measuring the expression of TM4SF4 gene in a blood sample, wherein the measurement of TM4SF4 gene expression specifically binds the protein encoded by the gene to the protein. It is related with the method including implementing by measuring using an antibody.

  Furthermore, the present invention relates to any one of the above-described methods for detecting liver cancer, which comprises preparing mononuclear cells from a blood sample and using the mononuclear cells as a sample.

  The present invention also relates to an oligonucleotide represented by the nucleotide sequence set forth in any one of SEQ ID NOs: 3 to 8 in the sequence listing.

  Furthermore, the present invention relates to a liver cancer comprising one or more of oligonucleotides and polynucleotides that specifically hybridize to mRNA or cDNA of TM4SF4 gene, and an antibody that specifically binds to a protein encoded by the gene. The present invention relates to a detection reagent kit.

  Furthermore, the present invention relates to a reagent kit for detecting liver cancer comprising one or more of oligonucleotides represented by the nucleotide sequence set forth in any one of SEQ ID NOs: 3 to 8 in the sequence listing.

  INDUSTRIAL APPLICABILITY According to the present invention, a method for detecting liver cancer, particularly hepatocellular carcinoma, comprising measuring the expression of TM4SF4 gene can be provided, and further a method for diagnosing liver cancer, particularly hepatocellular carcinoma, can be implemented. The method for detecting or diagnosing liver cancer according to the present invention is a useful method that can be carried out with very little invasiveness to a test subject because it can be performed using not only liver tissue samples but also blood samples as samples. .

  The present invention relates to a method for detecting liver cancer. The liver cancer detection method according to the present invention is characterized in that the TM4SF4 gene and / or a protein encoded by the gene (hereinafter sometimes referred to as TM4SF4) is measured as a liver cancer detection marker. That is, the present invention relates to a method for detecting liver cancer, comprising measuring TM4SF4 gene expression in a sample to be examined (hereinafter referred to as a test sample).

  The present inventors have found that the TM4SF4 gene is highly expressed in liver cancer tissues of human liver cancer patients compared to normal liver tissues, and that TM4SF4 mRNA is detected in peripheral blood mononuclear cells of human hepatocellular carcinoma patients. However, it was demonstrated that the mRNA was not detected in peripheral blood mononuclear cells of healthy individuals. Since TM4SF4 gene is highly expressed in liver cancer tissues and peripheral blood mononuclear cells of human liver cancer patients, the protein encoded by the gene is also detected in liver cancer tissues and peripheral blood mononuclear cells of liver cancer patients. Can do.

  From the above demonstration results, the present inventors carried out detection and diagnosis of liver cancer, preferably hepatocellular carcinoma, by measuring TM4SF4 gene and / or protein encoded by the gene as a marker for liver cancer detection in blood samples. I found out that I can do it.

  In the present invention, the term “gene” is used as a generic term to mean a full-length gene and a polynucleotide or oligonucleotide consisting of a part thereof, and includes DNA, RNA, mRNA, and cDNA, where the minimum The size is 2 nucleotides.

  “Gene expression” means that gene information is transcribed into mRNA, or is transcribed into mRNA and translated as an amino acid sequence of a protein.

  “Measuring gene expression” includes measuring the mRNA of the gene and measuring the protein encoded by the gene.

  “Measuring mRNA of a gene” includes detecting the presence or absence of mRNA and measuring the amount of mRNA.

  In the present invention, the term “protein” is used as a generic term to mean a full-length protein and a polynucleotide or oligopeptide comprising a part thereof, wherein the protein, polypeptide or oligopeptide has a minimum size of 2 amino acids. It is.

  “Measuring a protein” includes detecting the presence or absence of a protein and measuring its amount.

  The TM4SF4 gene has already been made public. For example, in a public database (GenBank etc.), the human TM4SF4 gene is registered as accession number NM_004617.2 (SEQ ID NO: 1). A protein encoded by the human TM4SF4 gene is disclosed in the accession number (SEQ ID NO: 2).

  The TM4SF4 gene is not limited to the polynucleotide consisting of the base sequence represented by SEQ ID NO: 1, but has the characteristics of having sequence homology with the polynucleotide and showing tissue expression equivalent to the polynucleotide Further, in addition to these characteristics, a polynucleotide encoding a protein having biological functions and structural characteristics equivalent to those of the protein encoded by the polynucleotide consisting of the base sequence represented by SEQ ID NO: 1 is included. The sequence homology is usually about 50% or more, preferably about 70% or more, more preferably about 80% or more, more preferably about 90% or more, and even more preferably about 95% or more of the entire base sequence. It is desirable.

  Tissue expression of the TM4SF4 gene has been observed in the liver, intestine and pancreas. In addition, the TM4SF4 gene is highly expressed in the liver cancer tissues of human liver cancer patients as described above compared to normal liver tissues. Thus, the TM4SF4 gene includes a polynucleotide having sequence homology with the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 and whose expression is observed in the liver, intestine and pancreas, particularly in the liver. The

  As a structural feature of the protein encoded by the TM4SF4 gene, it is known that the amino acid sequence has four transmembrane domains. Therefore, the TM4SF4 gene has sequence homology with the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, its expression is observed in the liver, intestine and pancreas, particularly the liver, and four transmembrane domains are present. Polynucleotides encoding the proteins they have are included.

  For the TM4SF4 gene, one or more, for example 1 to 100, preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 in the nucleotide sequence represented by SEQ ID NO: 1. Includes a polynucleotide represented by a nucleotide sequence having a mutation such as deletion, substitution, addition or insertion of 1 to 5 nucleotides. The degree of mutation and the position thereof are a polynucleotide having characteristics such that the polynucleotide having the mutation shows tissue expression equivalent to the polynucleotide consisting of the base sequence represented by SEQ ID NO: 1, and In addition, the polynucleotide is not particularly limited as long as it is a polynucleotide encoding a protein having biological functions and structural characteristics equivalent to those of the protein encoded by the polynucleotide consisting of the base sequence represented by SEQ ID NO: 1.

  The TM4SF4 gene includes a polynucleotide comprising a partial base sequence present in a specified region of the base sequence of the polynucleotide. A polynucleotide consisting of a partial base sequence present in a designated region of the base sequence of the polynucleotide is preferably 5 or more nucleotides, more preferably 10 or more nucleotides continuous in the region as a minimum unit. A polynucleotide consisting of 20 or more nucleotides is preferred. Specific examples of such a polynucleotide include a polynucleotide consisting of 609 to 840 consecutive nucleotides from the 232nd to the 840th, which is an open reading frame of a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1. .

  The TM4SF4 gene also includes a polynucleotide that hybridizes under stringent conditions to the polynucleotide or a part thereof. These polynucleotides do not have to be a polynucleotide having a complementary sequence as long as the polynucleotide hybridizes to the above-mentioned polynucleotide or a part thereof. Stringent hybridization conditions are well known. For example, the conditions described in the book (Sambrook et al., “Molecular Cloning, Laboratory Manual, Second Edition”, 1989, Cold Spring Harbor Laboratory, Section 11.45 “Conditions for Hybridization of Oligonucleotide Probes”, etc.), and is not particularly limited. Stringent conditions are determined by salt concentration, temperature, and other conditions. For example, the lower the salt concentration, the higher the temperature, the higher the stringency and the less likely it is to hybridize. The salt concentration is generally adjusted by adjusting the concentration of the SSC solution (NaCl + trisodium citrate), and stringent salt concentrations are, for example, about 250 mM or less NaCl and about 25 mM or less trisodium citrate. The stringent temperature is generally 15 to 25 ° C. lower than the melting temperature (Tm) of the complete hybrid, for example, about 30 ° C. or more. The temperature can be lowered by adding an organic solvent (eg, formamide) to the solution. Other conditions include the hybridization time, the concentration of a detergent (for example, SDS), the presence or absence of carrier DNA, and various stringencies can be set by combining these conditions. As a preferred example, hybridization is performed at a temperature of 42 ° C. under conditions of 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, 200 μg / ml denatured salmon sperm DNA. In addition, washing conditions after hybridization also affect stringency. This wash condition is also defined by salt concentration and temperature, and the stringency of the wash increases with decreasing salt concentration and increasing temperature. As a preferred example, washing is performed at a temperature of 68 ° C. under conditions of 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS.

  The protein encoded by the TM4SF4 gene is not limited to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, has sequence homology with the polypeptide, and is equivalent to the polynucleotide encoding the polypeptide. A polypeptide having characteristics such as being encoded by a polynucleotide exhibiting tissue expression, and in addition to these characteristics, it has biological functions and structural characteristics equivalent to those of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2. A polypeptide encoding a protein is included. The sequence homology is usually about 50% or more of the entire amino acid sequence, preferably about 70% or more, more preferably about 80% or more, more preferably about 90% or more, and even more preferably about 95% or more. It is desirable.

  Tissue expression of the gene encoding TM4SF4 has been observed in the liver, intestine and pancreas. In addition, the TM4SF4 gene is highly expressed in the liver cancer tissues of human liver cancer patients as described above compared to normal liver tissues. Thus, the protein encoded by the TM4SF4 gene has sequence homology with the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and its expression is observed in the liver, intestine and pancreas, particularly in the liver. Polypeptides encoded by the polynucleotides are included.

  As a structural feature of TM4SF4, it is known to have four transmembrane domains in its amino acid sequence. Therefore, the protein encoded by the TM4SF4 gene has a sequence homology with the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and is expressed by a polynucleotide whose expression is recognized in the liver, intestine and pancreas, particularly in the liver. Polypeptides that are encoded and have four transmembrane domains are included.

  The protein encoded by the TM4SF4 gene has one or more, for example 1 to 100, preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 1 in the amino acid sequence represented by SEQ ID NO: 2. Polypeptides represented by amino acid sequences having mutations such as deletion, substitution, addition or insertion of 10 amino acid residues, particularly preferably 1 to 5 amino acid residues are included. The degree of mutation and the position thereof, a polypeptide having such characteristics that the polypeptide having the mutation exhibits tissue expression equivalent to that of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, The polypeptide is not particularly limited as long as it is a polypeptide encoding a protein having biological functions and structural characteristics equivalent to those of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 in addition to the characteristics.

  The protein encoded by the TM4SF4 gene includes a polypeptide or oligopeptide consisting of a partial amino acid sequence present in a designated region of the amino acid sequence of the polypeptide. A polypeptide or oligopeptide consisting of a partial amino acid sequence present in a specified region of the amino acid sequence of the polypeptide is preferably 5 or more amino acid residues, more preferably 10 consecutive as the minimum unit. It is a polypeptide or oligopeptide consisting of the above amino acid residues, more preferably 20 or more amino acid residues.

  In the present invention, the term “cancer marker” is used interchangeably with the term tumor marker. A cancer marker means an indicator of cancerous growth or the presence or degree of cancer cells or cancer. In other words, a substance possessed by or produced by a cancer cell that exhibits a change that is significantly greater or less than that of a normal cell, or a substance that is mutated, or reacts with a cancer cell. Among substances produced by normal cells in the body, it means a substance that can determine cancer by detecting them. The cancer marker is preferably specific to the target cancer, but need not be completely specific, and can be detected in other diseases and organs as long as cancer can be detected. May be. Here, “specific to the target cancer” means that the target cancer can be detected with high sensitivity, but other cancers cannot be detected or the detection sensitivity is extremely low. To do. Further, “completely specific to the target cancer” means that only the target cancer can be detected and other cancers cannot be detected.

  In the present invention, the term “cancer” is used interchangeably with the term malignant tumor. Cancer refers to various malignant neoplasms, that is, tumors that exhibit metastatic or invasive properties with abnormal cell proliferation. Metastatic means the ability to form a tumor at a site that has migrated and moved from the primary site to a site that is anatomically separated. Infiltrating means the ability to enter the surrounding tissue from the site where the tumor was formed. A malignant neoplasm is generally a tissue mass that can be distinguished from normal tissue because the cells that make up it are morphologically different from normal cells and the sequence of the cells is different compared to normal tissues. Often formed.

  “Liver cancer” means a malignant neoplasm that develops in the liver, and includes primary liver cancer and liver cancer metastasized from primary liver cancer. Primary liver cancer means a malignant neoplasm that has developed in the liver and is not due to metastasis from other organs.

  “Hepatocellular carcinoma” is a type of primary liver cancer and is a malignant tumor with a poor prognosis that accounts for 90% or more of primary liver cancer.

  “Detecting liver cancer” means determining whether the test sample is a test sample derived from liver cancer.

  The present inventors have found that the TM4SF4 gene is highly expressed in liver cancer tissues of human liver cancer patients compared to normal liver tissues, and that TM4SF4 mRNA is detected in peripheral blood mononuclear cells of human hepatocellular carcinoma patients. However, it was demonstrated that the mRNA was not detected in peripheral blood mononuclear cells of healthy individuals. Since TM4SF4 gene is highly expressed in liver cancer tissues and peripheral blood mononuclear cells of human liver cancer patients, the protein encoded by the gene is also detected in liver cancer tissues and peripheral blood mononuclear cells of liver cancer patients. Can do.

  That is, it can be said that the mRNA of TM4SF4 gene and the protein encoded by the gene are increased in liver cancer tissue as compared with normal liver tissue. In addition, it can be said that the mRNA of TM4SF4 gene and the protein encoded by the gene are present at a detectable level in peripheral blood mononuclear cells of hepatocellular carcinoma patients. On the other hand, in healthy human peripheral blood mononuclear cells, it can be said that the mRNA of TM4SF4 gene and the protein encoded by the gene are present at a remarkably low level or undetectable level.

  Therefore, when an increased level of TM4SF4 gene mRNA and / or protein encoded by the gene is detected in the test sample, the test sample can be determined to be a sample derived from liver cancer. Alternatively, when mRNA of TM4SF4 gene and / or protein encoded by the gene is detected in cells prepared from a blood sample, more preferably in mononuclear cells prepared from a blood sample, the blood sample is derived from a patient with liver cancer. It can be determined to be a blood sample. The determination of whether the mRNA of the TM4SF4 gene and / or the protein encoded by the gene has increased can be carried out by comparison with a normal liver tissue or a blood sample derived from a healthy person. Alternatively, such a determination is made by measuring the standard value of TM4SF4 gene mRNA and / or the protein encoded by the gene detected in a normal liver tissue or a blood sample derived from a healthy person in advance. It can be implemented by comparison with.

  When a blood sample, preferably a cell prepared from a blood sample, more preferably a mononuclear cell prepared from a blood sample is used as a test sample, TM4SF4 mRNA is detected in peripheral blood mononuclear cells of patients with hepatocellular carcinoma, but healthy people In the peripheral blood mononuclear cells, the mRNA was not detected. Therefore, when the mRNA of the TM4SF4 gene and / or the protein encoded by the gene is detected, it is compared with a blood sample derived from a healthy person and the standard value. Instead, the blood sample can be determined to be a sample derived from liver cancer.

  The test sample is not particularly limited, and examples thereof include samples derived from living organisms such as blood, cells, urine, saliva, spinal fluid, biopsy tissue or autopsy material. Since it is known that the protein encoded by the TM4SF4 gene is a membrane protein having four transmembrane domains, it is appropriate that the test sample is a sample containing cells. The present inventors have demonstrated that TM4SF4 mRNA is detected in peripheral blood mononuclear cells of human hepatocellular carcinoma patients, but not in normal human peripheral blood mononuclear cells. From this, the test sample is preferably a blood sample, more preferably a cell prepared from a blood sample, more preferably a mononuclear cell prepared from a blood sample. “Mononuclear cells” are a group of mononuclear mesenchymal cells also referred to as mononuclear cells, and are widely distributed in connective tissue, lymphoid tissue and bloodstream. Mononuclear cells are classified into migrating mononuclear leukocytes represented by monocytes and lymphocytes, and mononuclear phagocytic cells represented by macrophages present in tissues. A biopsy tissue is also used as a test sample, but collection of the biopsy tissue is highly invasive to the patient and has a heavy burden. On the other hand, collection of a blood sample has the advantage that it is less invasive to the patient and can be easily performed.

  The measurement of TM4SF4 gene expression can be performed using a known gene detection method. Specific examples of gene detection methods include Southern blotting, Northern blotting, reverse transcription polymerase chain reaction (RT-PCR), Nucleic Acid Sequence-Based Amplification (NASBA), plaque hybridization, or colony hybridization. . Moreover, the measurement at the cell level using in situ RT-PCR, in situ hybridization, etc. can also be used. The method that can be used for detecting the target gene is not limited to the above method, and any known gene detection method can be used.

  In the gene detection method, a polynucleotide having a property as a probe or an oligonucleotide having a property as a primer is useful for detecting a gene, measuring the expression level of the gene, and performing gene amplification.

  An oligonucleotide having a property as a primer means one represented by a sequence unique to the gene that can specifically hybridize to the target gene and amplify the gene (hereinafter referred to as a primer). A polynucleotide having a property as a probe means one represented by a sequence unique to the gene that can specifically hybridize to the gene of interest (hereinafter referred to as a probe). Here, “a gene of interest can be specifically amplified” means that an amplification product can be formed by specific hybridization with the gene of interest, but an amplification product of other genes cannot be formed, or an amplification product Means less. In addition, “specifically hybridize to a target gene” means that a hybridization product with a target gene can be formed, but a hybridization product with other genes cannot be formed, or the amount of hybridization product is It means less.

  Detection of the TM4SF4 gene, measurement of the expression level of the gene, and amplification of the gene can be performed using a primer or probe that specifically hybridizes to the TM4SF4 gene. For example, detection of mRNA of the TM4SF4 gene, measurement of the amount of mRNA of the gene, and amplification of the amount of mRNA of the gene can be performed using a primer or probe that specifically hybridizes to the mRNA or cDNA of the gene.

  The primer for specifically amplifying the TM4SF4 gene may be any primer that can amplify at least a part of the gene. The probe that specifically hybridizes to the TM4SF4 gene may be any probe that can specifically hybridize to at least a part of the gene. Primers and probes can be appropriately designed based on the sequence information of the TM4SF4 gene according to a known method, and can be obtained by synthesis according to a conventional method. The primer generally has a base sequence length of preferably about 5 to 50 nucleotides, more preferably about 10 to 35 nucleotides, and even more preferably about 15 to 30 nucleotides. The probe generally has a base sequence length of 15 nucleotides or more, more preferably about 15 to 1000 nucleotides, and even more preferably about 50 to 500 nucleotides. As the probe, a labeled probe is usually used, but it may be unlabeled. Various methods for labeling a probe are known, and specific examples include a method using nick translation, random priming, or kinase treatment. Examples of suitable labeling substances include digoxigenin, radioisotopes, biotin, fluorescent substances, chemiluminescent substances, enzymes, tag peptides, and antibodies.

  As a primer for specifically amplifying the TM4SF4 gene, specifically, an oligonucleotide represented by the base sequence described in any one of SEQ ID NOs: 3 to 8 can be exemplified. These primers are preferably a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 3 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 4; A primer set composed of an oligonucleotide represented by the base sequence and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 6, and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 7 and SEQ ID NO: 8 It is suitable to be used in a primer set composed of an oligonucleotide represented by the base sequence described in 1. These oligonucleotides were created by designing and synthesizing based on the sequence information of the polynucleotide represented by the base sequence described in SEQ ID NO: 1.

  A gene can be detected by performing an amplification reaction or a hybridization reaction using an oligonucleotide having a property as a primer or a polynucleotide having a property as a probe, respectively, and detecting the amplification product or hybrid product.

  When performing an amplification reaction or a hybridization reaction, it is usually preferable to prepare a polynucleotide from a test sample. The polynucleotide to be prepared may be either RNA or DNA. Preparation of a polynucleotide from a test sample can be performed using a known method as appropriate. For example, when preparing RNA, a guanidine-cesium chloride ultracentrifugation method, a hot phenol method, or a guanidine thiocyanate-phenol-chloroform (AGPC) method is used. When preparing DNA, phenol extraction and ethanol precipitation are performed. Or a method using glass beads can be used. In addition, RNA and DNA can be prepared using commercially available RNA extraction reagents and RNA extraction kits, or DNA extraction reagents and DNA extraction kits. When RNA is prepared from a test sample, a reverse transcription reaction is further performed using the RNA as a template, cDNA can be prepared and used for an amplification reaction. Methods for preparing cDNA from RNA by reverse transcription are well known and can be carried out using commercially available RT-PCR kits and the like.

  The amplification reaction can be performed using a known amplification method capable of amplifying a desired polynucleotide. The amplification method is not particularly limited, but a DNA / RNA amplification method by polymerase chain reaction (PCR) can be suitably used. As long as PCR is a method using a primer capable of specifically amplifying a desired polynucleotide, any conventionally known method can be used. For example, RT-PCR, nested PCR, real-time PCR, and competitive PCR can be used, and various other variations of PCR used in the art can be applied. Other amplification methods include the LAMP (Loop-Mediated Isometric Amplification) method, the ICAN (Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids) method, and the RCA (Rolling Amplification method). it can.

  According to PCR, by using DNA as a template and a DNA polymerase repeatedly replicating only a specific site in the DNA base sequence, a polynucleotide comprising the base sequence of the specific site can be amplified. At this time, as a primer for the replication reaction, an oligonucleotide containing the base sequence at one end of the specific site and an oligonucleotide containing the base sequence at the other end are designed and created from the base sequence of the DNA. Use.

  According to RT-PCR, cDNA is prepared using reverse transcription reaction enzyme (RNA-dependent DNA polymerase) using RNA as a template, and then PCR is performed using the prepared cDNA as a template. A polynucleotide comprising a base sequence can be amplified.

  According to nested PCR, a polynucleotide amplified by PCR or RT-PCR is used as a template, and a replication reaction is performed by DNA polymerase using a primer set designed and created based on the base sequence of the amplified polynucleotide. Thus, a polynucleotide comprising the base sequence of a specific site of the amplified polynucleotide can be amplified. Nested PCR is used for the purpose of more reliably amplifying only the desired DNA.

According to real-time PCR, the initial amount of template DNA can be quantified based on the amplification rate by measuring the amplification by PCR over time using DNA as a template. Real-time PCR is performed using a fluorescent dye, and there are two methods. One is a method using a sequence-specific probe labeled with two kinds of fluorescent dyes that interfere with each other (TaqMan method), and the other is a dye that emits fluorescence only when intercalated into double-stranded DNA, such as ethidium. This method uses bromide, SYBR ^™ Green I, PicoGreen, or the like. In the TaqMan method, a probe that hybridizes to a specific region of a target gene, in which the 5 ′ end is labeled with a fluorescent dye (reporter) and the 3 ′ end is labeled with a quencher (quencher), is used. In the normal state of the probe, the fluorescence of the reporter is suppressed by the quencher. In a state where this fluorescent probe is hybridized to the target gene, PCR is performed from the outside using Taq DNA polymerase. As the extension reaction by Taq DNA polymerase proceeds, the exonuclease activity hydrolyzes the fluorescent probe from the 5 'end, and as a result, the reporter dye is released and emits fluorescence. The initial amount of template DNA can be quantified by measuring the intensity of this fluorescence over time. By using real-time PCR in combination with RT-PCR (real-time RT-PCR), a small amount of mRNA can be quantified.

  According to competitive PCR, a known amount of a competitive template RNA having a sequence complementary to a primer for detecting a desired mRNA and having a molecular weight or restriction enzyme cleavage site different from that of the target mRNA is added to the sample, and RT- The desired mRNA can be quantified by performing PCR after the PCR and comparing the amount of the desired amplification product derived from the mRNA and the amount of the amplification product derived from the competitive template template.

  Whether or not the amplification product obtained by the amplification reaction is a desired polynucleotide can be detected using a known method capable of specifically recognizing the amplification product. For example, it is possible to confirm whether or not a polynucleotide of a specific size is amplified using agarose gel electrophoresis or the like. Alternatively, the labeling substance is labeled on deoxynucleotide triphosphate (dNTP) incorporated into the amplification product during the amplification reaction, and the amplification product is detected by measuring the labeling substance of dNTP incorporated into the amplification product. be able to. Examples of the labeling substance include fluorescent substances such as fluorescein (FITC), sulforhodamine (SR) and tetramethylrhodamine (TRITC), and luminescent substances such as luciferin, and radioisotopes such as 32P, 35S and 121I. . The labeling method using these labeling substances and the labeling substance detection method are not particularly limited, and various conventionally known methods can be applied.

  The hybridization reaction is performed using a probe capable of hybridizing to a desired polynucleotide under conditions such that the probe specifically reacts with the polynucleotide. Conditions under which the probe specifically reacts with the polynucleotide, that is, stringent conditions are well known as described above and are not particularly limited.

  By labeling a labeling substance on the probe used for the hybridization reaction and measuring the labeling substance after the hybridization reaction, the hybridization product can be detected. Labeling substances include fluorescent substances such as fluorescein (FITC), sulforhodamine (SR) and tetramethylrhodamine (TRITC), luminescent substances such as luciferin, radioisotopes such as 32P, 35S and 121I, enzymes such as Examples include alkaline phosphatase and horseradish peroxidase, and biotin. The labeling method using these labeling substances and the labeling substance detection method are not particularly limited, and various conventionally known methods can be applied.

  A specific example of a method for detecting liver cancer, characterized by measuring TM4SF4 gene as a marker for liver cancer detection, will be described below. First, mononuclear cells are prepared from a blood sample. Mononuclear cells can be separated by a known method, for example, by a method such as specific gravity centrifugation. Next, RNA is extracted from the mononuclear cells, and cDNA is generated by reverse transcription using the obtained RNA as a template. Extraction of RNA and generation of cDNA by reverse transcription reaction can be performed according to the above-described polynucleotide preparation method and cDNA generation method. A TM4SF4 cDNA fragment polynucleotide is amplified and detected by nested PCR using the obtained cDNA as a template. Nested PCR is performed using the obtained cDNA as a template, for example, using a primer set composed of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 Then, using the 1st PCR product as a template, for example, 2nd PCR is performed using a primer set composed of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6. Can be implemented. The primer set is not limited thereto, and a primer set appropriately designed based on the base sequence of the TM4SF4 gene can be used. The 2nd PCR product is separated by electrophoresis, and a band corresponding to the size of the fragment polynucleotide of TM4SF4 cDNA that is expected to be amplified by nested PCR is detected using the above primer set. When such a band is detected, it can be determined that the TM4SF4 gene is expressed in the mononuclear cells prepared from the blood sample, and the blood sample that is confirmed to express the TM4SF4 gene is derived from liver cancer. It can be determined that this is a blood sample.

  The method for detecting liver cancer, which comprises measuring TM4SF4 gene as a marker for detecting liver cancer, can also be performed by extracting RNA from a test sample and using the RNA by real-time RT-PCR. As a primer set used here, the primer set comprised from the oligonucleotide which consists of an oligonucleotide which consists of a base sequence of sequence number 7 and a base sequence of sequence number 8 can be illustrated, for example. The primer set is not limited thereto, and a primer set appropriately designed based on the base sequence of the TM4SF4 gene can be used.

  The protein encoded by the TM4SF4 gene can be measured using a protein detection method or a quantification method according to conventional techniques in this field. For example, the target protein can be detected and quantified using an antibody (polyclonal or monoclonal antibody). Specifically, the above detection is possible by preparing a protein fraction from a test sample, performing immunoprecipitation using a specific antibody against the target protein, and analyzing the target protein by Western blotting or immunoblotting. is there. In addition, by using an antibody against the target protein, the target protein in paraffin or frozen tissue sections can be detected by immunohistochemical techniques. Alternatively, the target protein on the cell can be detected by cell staining using an antibody against the target protein. Specific examples of the method for detecting the target protein or a variant thereof include enzyme immunoassay (ELISA), sandwich method, radioimmunoassay (RIA), immunoradioassay (IRMA), immunoenzyme method (IEMA), Examples include competitive binding assays, immunofluorescence methods, Western blotting methods, immunoprecipitation methods, and protein chip analysis methods. As the antibody, a known antibody production method can be used by using a protein encoded by the TM4SF4 gene or a fragment protein of the protein as an antigen. Polyclonal antibodies can be obtained from the sera of animals that have been immunized by known antibody recovery methods. A preferred antibody recovery means is immunoaffinity chromatography. Monoclonal antibodies recover antibody-producing cells (for example, spleen or lymph node-derived lymphocytes) from animals that have been subjected to immunization, and are known per se proliferative cells (for example, P3-X63-Ag8 strain) Can be produced by introducing means for transformation into a myeloma strain.

  The detection method according to the present invention is effective for detection of liver cancer, preferably hepatocellular carcinoma. The detection method according to the present invention can be used to diagnose liver cancer, preferably hepatocellular carcinoma. Therefore, a method for diagnosing liver cancer, preferably hepatocellular carcinoma, using the detection method according to the present invention is also included in the scope of the present invention. When diagnosing liver cancer, the detection method according to the present invention is performed, and conventional markers such as AFP, AFP L3 fraction, and PIVKA-II are measured, blood tests such as complete blood count (Complete) are performed. Blood Count, CBC), liver function test, kidney function test, and the like are performed, and liver cancer can be determined by comparing with the results. In addition, if there is an isolated specimen or cytology, liver cancer can be determined by comparing with the results.

  The present invention also relates to reagents and reagent kits used for detection of liver cancer, preferably hepatocellular carcinoma.

  As a reagent according to the present invention, an oligonucleotide having a property as a primer capable of specifically amplifying a TM4SF4 gene or a fragment polynucleotide of the gene, a probe specifically hybridizing to the TM4SF4 gene or a fragment polynucleotide of the gene And an antibody that specifically binds to a protein encoded by the TM4SF4 gene. For example, examples of the reagent according to the present invention include oligonucleotides and polynucleotides that specifically hybridize to TM4SF4 gene mRNA or cDNA. Specifically, for example, an oligonucleotide represented by the base sequence described in any one of SEQ ID NOs: 3 to 8 can be exemplified as the reagent according to the present invention. The reagent according to the present invention can be pre-labeled with a labeling substance. Examples of the labeling substance include the aforementioned proteins and radioisotopes. The reagent according to the present invention may contain substances such as buffers, salts, stabilizers, and / or preservatives. In formulating, a formulation unit known per se according to the properties of each substance may be introduced. The reagent according to the present invention can be used in the method for detecting liver cancer according to the present invention and the method for diagnosing liver cancer using the detection method. Here, “specifically binds to a protein encoded by the TM4SF4 gene” means that it binds to the protein but does not bind or weakly binds to other proteins.

  As a reagent kit according to the present invention, an oligonucleotide having a property as a primer capable of specifically amplifying a TM4SF4 gene or a fragment polynucleotide of the gene, a probe that specifically hybridizes to the TM4SF4 gene or a fragment polynucleotide of the gene And a reagent kit comprising at least one of a polynucleotide having the following properties and an antibody that specifically binds to a protein encoded by the TM4SF4 gene. For example, as a reagent kit according to the present invention, at least one of an oligonucleotide and a polynucleotide that specifically hybridize to mRNA or cDNA of TM4SF4 gene, and an antibody that specifically binds to a protein encoded by the gene A reagent kit containing one can be exemplified. Specifically, for example, as a reagent kit according to the present invention, a reagent kit containing an oligonucleotide represented by the base sequence described in any one of SEQ ID NOs: 3 to 8 can be exemplified. Preferably, a set of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 3 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 4; an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 5 And an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 6, and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 7 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 8. A reagent kit containing any one set selected from these sets can be exemplified. More preferably, it is represented by a set of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 3 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 4 and a base sequence set forth in SEQ ID NO: 5. A reagent kit including a set of an oligonucleotide and an oligonucleotide represented by the base sequence described in SEQ ID NO: 6 can be exemplified. The reagent kit according to the present invention contains other substances useful for carrying out the detection method of the present invention, such as a labeled substance, a detection agent for the labeled substance, a reaction diluent, a standard antibody, a buffer solution, a cleaning agent, a reaction stop solution, and the like. Can be included. Examples of the labeling substance include the aforementioned proteins and radioisotopes. The labeling substance may be added to the polynucleotide, oligonucleotide, or antibody in advance. The reagent kit according to the present invention can be used in the method for detecting liver cancer according to the present invention and the method for diagnosing liver cancer using the detection method. Further, the reagent kit according to the present invention can be used as a determination kit and a diagnosis kit for liver cancer, preferably hepatocellular carcinoma.

  The present invention also relates to a DNA chip used for detection of liver cancer, preferably hepatocellular carcinoma. In the present invention, the term DNA chip is used interchangeably with the term DNA array. The DNA chip according to the present invention can be produced by immobilizing a polynucleotide capable of hybridizing with TM4SF4 mRNA on a substrate such as silicon or glass. DNA can be immobilized on a substrate using a well-known method (Shena M. et al., “Science”, 1995, Vol. 270, No. 5235, p. 467-470).

  Gene groups that change during the carcinogenesis process in a mouse liver carcinogenesis model were screened using a DNA microarray.

Specifically, c-myc / TGF-α double transgenic mice (Murakami H. et. Al., “Cancer Research”, 1993, Vol. 53, No. 8, p. 1719-1723), 20 of the liver tumors developed in this model mouse were collected, and RNA was extracted with TRIZOL ^™ . At this time, the tumor was cut at the maximum cleavage plane to prepare a tissue section having a thickness of about 3 mm, and a hematoxylin-eosin stained specimen (HE specimen) was prepared.

  Ten cancers and adenomas confirmed in the HE specimen were selected, and 20 μg of total RNA (tRNA) was prepared from each and used as a sample. Samples prepared from cancer and adenomas were labeled with Cy5 and Cy3, respectively. Labeling was performed using an Atlas glass labeling kit (Clontech). Labeled samples were hybridized on ATC 2.6K mouse Onco-chip (ATC, NCI) and incubated at 37 ° C. for 48 hours. Hybridization was performed according to the attached protocol.

  As a result, it was found that TM4SF4 mRNA was highly expressed in the liver cancer tissue generated in the mouse liver carcinogenesis model as compared with the liver adenoma tissue.

  TM4SF4 mRNA expression was compared between liver tumors generated in a mouse liver carcinogenesis model and liver tissues of normal mice. In this examination, TM4SF4 expression in each tissue was detected by an in situ hybridization method (sometimes abbreviated as ISH), and the copy number of TM4SF4 mRNA was measured by real-time RT-PCR.

  Specifically, TM4SF4 mRNA expression was detected in liver dysplasia, hepatic adenoma and liver cancer, and wild type mice (sometimes abbreviated as WT) generated in c-myc / TGF-α double transgenic mice. A comparative study was performed using liver tissue.

Detection of tissue expression by in situ hybridization was performed by Northern blotting using a tissue section prepared from each tissue and a probe prepared as described below. In order to prepare a probe, first, a mouse Image: 423204 clone was purchased from ResGen, and the clone was sequenced using T3 and T7 primers to confirm that the TM4SF4 gene was contained. Using this clone as a template, PCR was performed using a primer set consisting of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 9 of the Sequence Listing and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 10, and a 279 bp PCR The product was obtained. The PCR product was subjected to electrophoresis, the obtained band was cut out and purified, and the obtained polynucleotide was used as a probe (hereinafter referred to as mouse TM4SF4 probe). RNA was extracted from each tissue with TRIZOL ^™ , 15 μl thereof was subjected to electrophoresis, transferred to a membrane, and then hybridized with a mouse TM4SF4 probe labeled with 32P.

Measurement of the copy number of TM4SF4 mRNA by real-time RT-PCR was performed using the same RNA as that used for the detection of the tissue expression. First, using SuperScript ^™ III First-Strand Synthesis System for PCR (manufactured by Invitrogen), cDNA was generated by reverse transcription reaction using 1 μg of tRNA as a template according to the attached protocol. Subsequently, real-time PCR was performed using ABI PRISM 7700HT (manufactured by Applied Biosystems). As a primer set, a primer set composed of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 12 was used. This primer set amplifies a 102 bp mouse TM4SF4 cDNA fragment.

  As a result of the above examination, it was found that the copy number of TM4SF4 mRNA was significantly increased in the liver cancer tissue generated in the mouse liver carcinogenesis model as compared with the dysplasia, hepatic adenoma tissue and the liver tissue of the wild type mouse. (FIG. 1). In addition, it was found that TM4SF4 mRNA is expressed in Gleason and the central venous region in normal liver tissue, and its expression is increased in liver cancer tissue.

  From the above results, the TM4SF4 gene was determined to be a gene whose expression increases in the process of carcinogenesis from liver adenoma to liver cancer.

The expression of TM4SF4 mRNA in human hepatocellular carcinoma liver cancer tissue was compared with the expression in liver tissue around the liver cancer tissue and normal liver tissue. As tissue samples, 57 cases of a human liver cancer tissue library (Laboratory of Experimental Carcinogenesis) were used. RNA was extracted from each tissue with TRIZOL ^™, and real-time RT-PCR was performed using ABI PRISM 7700HT (manufactured by Applied Biosystems). As a primer set, a primer set composed of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 7 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 8 was used. This primer set amplifies a 102 bp TM4SF4 cDNA fragment.

  As a result of the above examination, it was found that the expression of TM4SF4 mRNA in human hepatocellular carcinoma tissues of 57 cases was significantly increased as compared with the liver tissues around the cancer tissues and normal liver tissues (FIG. 2). In 45.3% of the 57 cases, the expression of TM4SF4 mRNA in the cancer tissue was more than twice as high as that in the liver tissue around the cancer tissue.

  Thus, also in human liver cancer cases, it was revealed that the expression of TM4SF4 gene in liver cancer tissues was remarkably higher than that of liver tissues around the liver cancer tissues and normal liver tissues. From these results, the present inventors believe that the TM4SF4 gene and the protein encoded by the gene can be used for detection of liver cancer as a liver cancer marker.

  We compared the expression of TM4SF4 gene in blood samples collected from primary liver cancer cases, metastatic liver cancer cases, and healthy subjects. Blood samples were collected after obtaining informed consent. The subjects were 19 cases of hepatocellular carcinoma as hepatocellular carcinoma cases, 2 cases of liver metastasis of pancreatic cancer as cases of metastatic liver cancer, and 2 cases of healthy subjects.

  Examination of TM4SF4 gene expression was performed by detecting TM4SF4 mRNA expression in mononuclear cell samples separated from blood samples.

1. Separation of mononuclear cell layer First, 10 ml of venous blood (hereinafter, sometimes referred to as peripheral blood) was collected with an EDTA (ethyleniamine tetraacetate) blood collection tube and stored frozen. Within 8 hours after collection, the mononuclear cell layer was separated from the blood sample by the concentration gradient method. Specifically, 25 ml of phosphate buffered saline (PBS) was added to the collected blood sample, diluted, and overlaid on 15 ml of Ficoll Paque ^™ (Amersham Biosciences) added to a 50 ml centrifuge tube. Thereafter, centrifugation was performed at 1200 g and 4 ° C. for 30 minutes. Intermediate layer cells were harvested and transferred to a new 50 ml centrifuge tube and washed three times with PBS. For washing, first add 25 ml of PBS and centrifuge at 1200 g at 4 ° C. for 10 minutes, then add 25 ml of PBS to the pellet and centrifuge at 1200 g at 4 ° C. for 10 minutes, and finally add 1 ml of PBS and The nuclei were suspended, transferred to a 2 ml centrifuge tube, and centrifuged at 1200 g at 4 ° C. for 2 minutes. Thereafter, the supernatant was discarded and stored frozen at −80 ° C. as a pellet only.

2. Extraction of RNA tRNA was extracted from the cryopreserved pellet. Extraction of tRNA was performed using a tRNA extraction kit, RNeasyMini ^™ (Qiagen) according to the attached protocol. Thereafter, DNase treatment (manufactured by Qiagen) was performed using RNase-Free DNase Set. The concentration and quality of the obtained RNA were measured using an absorptiometer.

3. Reverse Transcriptase Reaction, Nested RT-PCR Reaction cDNA was generated by reverse transcription reaction using 1 μg of tRNA as a template. The reverse transcription reaction was performed using Super Script ^™ III First-Strand Synthesis System for RT PCR (manufactured by Invitrogen) according to the attached protocol.

TM4SF4 cDNA was amplified and detected by RT-nested-PCR using the obtained cDNA as a template. RT-nested-PCR was performed according to the attached protocol using Platinum ^™ PCR SuperMix High Fidelity (manufactured by Invitrogen).

  Specifically, first, 2 μl of cDNA was used as a template, and a primer set composed of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 was used as a primer set. The annealing temperature was 55 ° C., and PCR (1st PCR) was performed in 35 cycles. This primer set amplifies a TM4SF4 cDNA fragment corresponding to 350 bp composed of the 391st to 740th nucleotides of the TM4SF4 gene (SEQ ID NO: 1).

  Next, using 1 μl of the 1st PCR product as a template, a primer set consisting of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6 was used as a primer set, PCR (2nd PCR) was performed in 35 cycles at 55 ° C. With this primer set, a TM4SF4 cDNA fragment corresponding to 163 bp consisting of nucleotides 428 to 595 of the TM4SF4 gene (SEQ ID NO: 1) is amplified.

The β-actin gene was employed as an internal control and its expression was detected. Detection of β-actin gene expression was performed by amplifying β-actin cDNA by PCR using the generated cDNA as a template. PCR was performed using Super Script ^™ III First-Strand Synthesis System for RT PCR (manufactured by Invitrogen) according to the attached protocol. Specifically, annealing is performed using 1 μl of the above cDNA as a template and a primer set composed of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 13 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14 as a primer set. PCR was performed in 30 cycles at a temperature of 63 ° C. This primer set amplifies a 234 bp β-actin cDNA fragment.

  The results are summarized in Table 1, and representative results among the results obtained by RT-nested-PCR are shown in FIG. In Table 1 and FIG. 3, HCC, Liver Metastasis, and Control indicate a hepatocellular carcinoma case, a pancreatic cancer liver metastasis case, and a healthy person, respectively.

  As shown in Table 1, TM4SF4 mRNA was detected in a mononuclear cell sample prepared from peripheral blood in 18 out of 19 cases of hepatocellular carcinoma. On the other hand, TM4SF4 mRNA was detected in a mononuclear cell sample prepared from peripheral blood in 1 of 2 cases of liver metastasis of pancreatic cancer. In contrast, TM4SF4 mRNA was not detected in mononuclear cell samples prepared from peripheral blood derived from healthy individuals.

  Thus, it was found that TM4SF4 mRNA is not detected in a mononuclear cell sample prepared from peripheral blood derived from a healthy person but detected in a mononuclear cell sample prepared from peripheral blood derived from a liver cancer case. . Since the expression level of β-actin gene in each mononuclear cell sample derived from a liver cancer case and a healthy person is approximately the same, the expression of TM4SF4 mRNA in a mononuclear cell sample derived from a hepatocellular carcinoma case is It is clear that this is not an artificial result due to differences in quantity.

  Based on the above results and the expression specificity of the TM4SF4 gene in the liver and pancreas, the present inventors believe that the TM4SF4 gene and the protein encoded by the gene can be used as a liver cancer marker. Moreover, since the TM4SF4 gene and the protein encoded by the gene can be detected using a blood sample, it is simple and less invasive to patients, and is highly useful for detection and diagnosis of liver cancer.

  According to the present invention, a method for detecting or diagnosing liver cancer, particularly hepatocellular carcinoma, characterized by using TM4SF4 gene or a protein encoded by the gene as a marker and further detecting the marker can be carried out.

  Since the measurement of this marker can be performed using not only a liver tissue sample but also a blood sample as a test sample, the invasiveness to a patient is small and can be easily performed, and is highly useful. Furthermore, by measuring this marker, it is possible to determine liver cancer, particularly hepatocellular carcinoma, which has been difficult to determine in the past, more accurately and easily.

  Thus, the present invention is a method for detecting or diagnosing liver cancer, particularly hepatocellular carcinoma, characterized by measuring the TM4SF4 gene and / or the protein encoded by the gene, and using a blood sample It is very useful in the pharmaceutical industry in that it can provide a possible method.

TM4SF4 gene expression in liver cancer tissues (indicated as Carcinoma) generated in a mouse liver carcinogenesis model, dysplastic foci (indicated as Dyplastic), hepatic adenoma tissues (indicated as Adeno), and liver tissues of wild-type mice (indicated as WT) It is a figure which showed having increased significantly compared with (). TM4SF4 mRNA was quantified by real-time RT-PCR, and the amount of mRNA in each tissue was indicated by the number of mRNA copies. The vertical axis represents the TM4SF4 mRNA copy number (indicated as “Absolute copy number of TM4SF4” in the figure). In the figure, the numbers shown at the top of each column indicate the number of samples. TM4SF4 gene expression is significant in 57 cases of human hepatocellular carcinoma tissue (labeled Tumor) compared to surrounding normal liver tissue (labeled Normal) and liver tissue surrounding the cancer tissue (labeled Non-tumor) It is a figure which shows having increased. TM4SF4 mRNA was quantified by real-time RT-PCR, and the amount of mRNA in each tissue was indicated by the number of mRNA copies. The vertical axis represents the TM4SF4 mRNA copy number (indicated as “Absolute copy number of TM4SF4” in the figure). In the figure, the numbers shown at the top of each column indicate the number of samples. TM4SF4 mRNA was detected in a mononuclear cell sample prepared from peripheral blood of a hepatocellular carcinoma case (indicated as HCC), but detected in a mononuclear cell sample prepared from peripheral blood from a healthy person (indicated as Control). It is a figure which shows that it was not done. On the other hand, TM4SF4 mRNA was detected in a mononuclear cell sample prepared from peripheral blood of one of two cases of pancreatic cancer liver metastasis (indicated as Liver metastasis). TM4SF4 mRNA was detected by RT-nested-PCR. When the expression level of β-actin gene was measured in the same manner as an internal control, the expression level was almost the same in all samples.

SEQ ID NO: 1: human TM4SF4 gene.
SEQ ID NO: 2: protein encoded by the human TM4SF4 gene (SEQ ID NO: 1).
SEQ ID NO: 3: Oligonucleotide designed for human TM4SF4 gene amplification primer.
SEQ ID NO: 4: Oligonucleotide designed for human TM4SF4 gene amplification primer.
SEQ ID NO: 5: oligonucleotide designed for human TM4SF4 gene amplification primer.
SEQ ID NO: 6: oligonucleotide designed for human TM4SF4 gene amplification primer.
SEQ ID NO: 7: oligonucleotide designed for human TM4SF4 gene amplification primer.
SEQ ID NO: 8: oligonucleotide designed for human TM4SF4 gene amplification primer
SEQ ID NO: 9: oligonucleotide designed for mouse TM4SF4 gene amplification primer.
SEQ ID NO: 10: oligonucleotide designed for mouse TM4SF4 gene amplification primer
SEQ ID NO: 11: oligonucleotide designed for mouse TM4SF4 gene amplification primer.
SEQ ID NO: 12: oligonucleotide designed for mouse TM4SF4 gene amplification primer
SEQ ID NO: 13: oligonucleotide designed for human β-actin amplification primer.
SEQ ID NO: 14: oligonucleotide designed for human β-actin amplification primer.

Claims (10)

A method for detecting liver cancer, comprising measuring the expression of transmembrane 4 superfamily 4 (TM4SF4) gene in a blood sample. The method for detecting liver cancer according to claim 1, comprising measuring TM4SF4 gene expression using a primer or probe that specifically hybridizes to mRNA or cDNA of the gene. The method for detecting liver cancer according to claim 1, comprising measuring TM4SF4 gene expression using at least one of the following primer sets:
(1) A primer set composed of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 3 in the sequence listing and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 4.
(2) A primer set composed of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 5 in the sequence listing and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 6; and (3) a sequence set forth in the sequence listing. A primer set composed of an oligonucleotide represented by the base sequence described in No. 7 and an oligonucleotide represented by the base sequence described in SEQ ID No. 8. The method for detecting liver cancer according to claim 1, wherein the measurement of TM4SF4 gene expression comprises the following steps:
(I) A primer set comprising a TM4SF4 gene cDNA fragment polynucleotide comprising an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing and an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 4 A step of forming a primary amplification product by amplification by
(Ii) A primer set comprising a fragment polynucleotide of the primary amplification product consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 5 in the sequence listing and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 6 A step of forming a secondary amplification product by amplification by (iii), and a step of detecting the secondary amplification product. The method for detecting liver cancer according to claim 1, comprising measuring the expression of the TM4SF4 gene by measuring a protein encoded by the gene. The method for detecting liver cancer according to claim 5, comprising measuring a protein encoded by the TM4SF4 gene using an antibody that specifically binds to the protein. The method for detecting liver cancer according to any one of claims 1 to 6, wherein mononuclear cells are prepared from a blood sample, and the mononuclear cells are used as a sample. An oligonucleotide represented by the base sequence set forth in any one of SEQ ID NOs: 3 to 8 in the Sequence Listing. Transmembrane 4 superfamily 4 (TM4SF4) Oligonucleotides and polynucleotides that specifically hybridize to mRNA or cDNA of the gene, and antibodies that specifically bind to the protein encoded by the gene A reagent kit for detecting liver cancer comprising two or more. A reagent kit for detecting liver cancer comprising one or more of oligonucleotides represented by the nucleotide sequence set forth in any one of SEQ ID NOs: 3 to 8 in the sequence listing.