Hepatocellular carcinoma-specific biomarker

Abstract

The present invention relates to using genes that express specific alterations for hepatocellular carcinoma as biomarkers for detecting and diagnosing hepatocellular carcinoma cells. As biomarkers of the present invention, hyaluronan-mediated motility receptor (HMMR), neurexophilin 4 (NXPH4), paired-like homeodomain 1 (PITX1), thrombospondin 4 (THBS4), or ubiquitin-conjugating enzyme E2T (UBE2T) specifically alter their expression for hepatocellular carcinoma. Therefore, they have the following effects: they can be used as hepatocellular carcinoma-specific markers, and if they are used separately alone, or in combination with alpha-fetoprotein, or in other combinations, hepatocellular carcinoma can be diagnosed more specifically and accurately.

Description

Technical Field

[0001] The present invention relates to a liver cancer-specific biomarker, and more particularly, to a gene whose expression is specifically altered for hepatocellular carcinoma and which is used as a biomarker for detecting and diagnosing liver cancer cells. Background Art

[0002] Among cancers, liver cancer is known as one of the most lethal cancers in the world. It is reported that particularly in Asia and sub-Saharan Africa, more than about 500,000 people die from liver cancer every year. Liver cancer can be roughly divided into primary liver cancer (hepatocellular carcinoma) caused by liver cells themselves and metastatic liver cancer caused by cancer metastasis from other tissues to the liver. Among them, more than about 90% of liver cancers are primary liver cancers.

[0003] Hepatocellular carcinoma (HCC) is the fifth most common tumor in the world, with 500,000 people dying from liver cancer every year (Okuda 2000). In the past 20 years, the survival rate of patients with liver cancer cells has not improved, and its incidence is approximately equal to the mortality rate (Marrero, Fontana et al 2005). Chronic hepatitis caused by hepatitis B virus or hepatitis C virus infection and exposure to carcinogens such as aflatoxin B1 is known to be the main risk factor for liver cancer cells (Thorgeirsson and Grisham 2002). Although it has been reported that changes in cell cycle regulators that progress from the cell cycle mechanism to the G1 phase are related to the formation of liver cancer (Hui et al, Hepatogasteroenterology 45:1635-1642, 1998), the intracellular molecular mechanisms related to the onset and progression of liver cancer are still unclear. According to previous studies, when protooncogenes such as various growth factor genes mutate into oncogenes and are overexpressed or overactive for various reasons, or tumor suppressor genes such as Rb protein or p53 protein mutate and are underexpressed or lose their function for various reasons, it has been reported that the onset and progression of various liver cancers including liver cancer will be caused. In addition, genetic alterations such as DNA mutations and gene expression have been confirmed in the tissues of liver cancer patients (Park et al, Cancer Res 59:307-310, 1999; Bjersing et al, J Intern Med 234:339-340, 1993; Tsopanomichalou et al, Liver 19:305-311, 1999; Kusano et al, Hepatology29:1858-1862, 1999; Keck et al, Cancer Genet Cytogenet 111:37-44, 1999). In recent years, it has been recognized that the onset and progression of most cancers including liver cancer are not caused by a specific few genes, but by the complex interaction of various genes related to the cell cycle, signal transduction, etc. Therefore, it is necessary to conduct a comprehensive study of various genes or proteins, rather than only focusing on the expression or function of a single gene or protein.

[0004] On the other hand, a biomarker detection method that can accurately detect liver cancer at an early stage in the general population has not been developed. The non-invasive early liver cancer detection method used for diagnosing high-risk populations is serum alpha-fetoprotein detection. When it was developed, 20 ng / mL was proposed as the reference value that could simultaneously achieve excellent sensitivity and specificity of alpha-fetoprotein (AFP). However, in this case, the sensitivity was only 60%. When diagnosing liver cancer according to the liver cancer diagnosis guidelines of the international association with a standard of 200 ng / mL, although the specificity increased, the sensitivity was only 22%. Based on the results of previous studies, alpha-fetoprotein is generally known to have a sensitivity of about 66% and a specificity of 82%, so there are limitations in diagnosing all liver cancer patients. In addition, although diagnostic criteria have not been established, serum markers that are helpful for liver cancer diagnosis include descarboxyprothrombin (DCP), prothrombin induced by vitamin K absence II (PIVKA-II), the distribution of glycosylated alpha-fetoprotein and total alpha-fetoprotein (L3 fraction), alpha fucosidase, glypican 3, heat shock protein (HSP)-70, etc. However, most of them have the meaning of a prognostic factor, and when used alone, their accuracy is low, so they cannot be used for screening detection, and the early diagnosis of liver cancer is judged to have reached its limit. The patients diagnosed at the curative treatment stage where actual surgery or high-frequency thermotherapy can be performed are limited to about 30% of all liver cancer patients. Summary of the Invention

[0005] Technical Problem

[0006] The object of the present invention is to develop a new diagnostic marker with improved specificity and sensitivity, which can maximize early diagnosis at the stage where liver cancer can be cured.

[0007] Technical Means

[0008] To achieve the above object, the present invention provides a biomarker for diagnosing liver cancer.

[0009] Furthermore, the present invention provides a composition for diagnosing liver cancer.

[0010] Furthermore, the present invention provides a liver cancer diagnostic kit.

[0011] Meanwhile, the present invention provides a method for providing information required for diagnosing liver cancer.

[0012] Technical Effects

[0013] According to the present invention, the hyaluronan-mediated motility receptor (HMMR), neurexophilin 4 (NXPH4), paired-like homeodomain 1 (PITX1), thrombospondin 4 (THBS4), or ubiquitin-conjugating enzyme E2T (UBE2T), which are biomarkers of the present invention, specifically change their expression for hepatocellular carcinoma, and thus have the following effects: They can be used as hepatocellular carcinoma-specific markers, and if they are used separately alone, or in combination with alpha-fetoprotein, or in other combinations, hepatocellular carcinoma can be diagnosed more specifically and accurately. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram showing the derivation process of the hepatocellular carcinoma-specific marker of the present invention.

[0015] Figure 2 It is a diagram showing the derivation process of 2502 pre-cancer genomes overexpressed only in hepatocellular carcinoma.

[0016] Figure 3 It is a diagram of the result of hierarchical clustering analysis of 737 genes overexpressed in both of two databases by analyzing The Cancer Genome Atlas hepatocellular carcinoma (TCGA_LIHC) data and the Gene Expression Omnibus (GEO) database.

[0017] Figures 4a - 4d It is a diagram for confirming 10 candidate marker genes overexpressed in both the GSE114564 data cohort and GSE6764.

[0018] Figures 5a - 5d It shows the results of separately analyzing the expression levels of 10 marker genes in non-tumor tissues and tumor tissues of hepatocellular carcinoma patients from the TCGA_LIHC data set and the ICGC_LIRI data set.

[0019] Figures 6a - 6b It shows the results of comparative analysis of the expression levels of the above 10 marker genes using the GSE77314 data set.

[0020] Figure 7It is a graph confirming the alpha-fetoprotein value as a cancer marker for liver cancer cells in an independent cohort of liver disease patients (771 specimens from 100 patients).

[0021] Figures 8a - 8b It is the result of analyzing the expression levels of 10 selected marker genes by enzyme-linked immunosorbent assay (ELISA).

[0022] Figures 9a - 9b It is the result of performing receiver operating characteristic (ROC) curve analysis using the ELISA values of 10 marker genes.

[0023] Figure 10 It is a graph confirming the alpha-fetoprotein value as a cancer marker for liver cancer cells in an independent cohort of liver disease patients (1148 specimens from 279 patients).

[0024] Figures 11a - 11b The result of analyzing the expression levels of 10 marker genes by enzyme-linked immunosorbent assay.

[0025] Figures 12a - 12b It is the result of performing receiver operating characteristic curve analysis using the ELISA values of the hyaluronan-mediated motility receptor, neuropilin 4, paired homeodomain 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T of the marker genes.

[0026] Figures 13a - 13g It is a graph analyzing and confirming the sensitivity, specificity, and accuracy of the hyaluronan-mediated motility receptor, neuropilin 4, paired homeodomain 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T in a cohort validated using alpha-fetoprotein by enzyme-linked immunosorbent assay.

[0027] Figures 14a - 14d It is a comparison graph of the diagnostic efficacy of hepatocellular carcinoma according to the combination of two markers among alpha-fetoprotein, hyaluronan-mediated motility receptor, neuropilin 4, paired homeodomain 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T (the combination of alpha-fetoprotein and hyaluronan-mediated motility receptor, neuropilin 4, paired homeodomain 1, thrombospondin 4, or ubiquitin-conjugating enzyme E2T; or the combination of two markers among hyaluronan-mediated motility receptor, neuropilin 4, paired homeodomain 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T) or the combination of three markers (the combination of alpha-fetoprotein and two markers among hyaluronan-mediated motility receptor, neuropilin 4, paired homeodomain 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T; or the combination of three markers among hyaluronan-mediated motility receptor, neuropilin 4, paired homeodomain 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T).

[0028] Optimal Embodiment

[0029] Hereinafter, the present invention will be described in detail by way of examples of the present invention with reference to the accompanying drawings. However, the following examples are presented as illustrations of the present invention, and when it is judged that a detailed description of techniques or structures known to those of ordinary skill in the technical field to which the present invention pertains may unnecessarily obscure the gist of the present invention, the detailed description thereof may be omitted, and the present invention is not limited thereto. Within the scope of the claims of the following invention and the equivalents construed therefrom, the present invention can be variously modified and applied.

[0030] Moreover, the terminology used in this specification is the terminology used to appropriately express the preferred embodiments of the present invention, and it may vary according to the intention of the user or operator or the convention in the field to which the present invention pertains, etc. Therefore, these terms should be defined based on the content throughout the specification. Throughout the specification, when it is stated that a certain part "comprises" a certain structural element, unless there is a particularly contrary statement, this does not mean excluding other structural elements, but rather means that other structural elements may also be included.

[0031] Unless otherwise indicated, nucleic acids are recorded in the 5'→3' direction from left to right. The numerical ranges exemplified in the specification include the numbers used to define the range, and include each integer or any non-integer fraction within the defined range.

[0032] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains. Any methods and materials similar or equivalent to those described in this specification can be used in the practice for testing the present invention, but the preferred materials and methods are described herein.

[0033] In the present invention, the term "subject" or "patient" refers to any individual in need of treatment, including humans, apes, monkeys, cows, dogs, guinea pigs, rabbits, chickens, insects, etc. Moreover, the subjects include any subjects participating in a clinical research trial who do not show any clinical manifestations of a disease or subjects participating in a mechanical study or subjects used as a control group.

[0034] In the present invention, the term "specimen (sample)" refers to a biological specimen obtained from a subject or patient. The source of the biological specimen can be fresh, frozen, and / or preserved organ or tissue samples or solid tissues generated from biopsies or primers; blood or any blood component; cells at any time point during pregnancy or development of the subject. In one embodiment of the present invention, blood or any blood component is used as the specimen.

[0035] Unless otherwise defined, all technical terms used in the present invention are used with the same meanings as those commonly understood by those of ordinary skill in the technical field to which the present invention pertains in the relevant fields of the present invention. And, although preferred methods or specimens are described in the present invention, similar or equivalent ones are also included within the scope of the present invention. The content of all publications cited as references in this specification is incorporated into the present invention.

[0036] In one aspect, the present invention relates to biomarkers for diagnosing liver cancer, which comprise one or more genes selected from the group consisting of alpha-fetoprotein, hyaluronan-mediated motility receptor (HMMR), neurexophilin 4 (NXPH4), paired-like homeodomain 1 (PITX1), thrombospondin 4 (THBS4), and ubiquitin-conjugating enzyme E2T (UBE2T) that exhibit specific expression changes for liver cancer, or proteins expressed from the above genes. In one embodiment of the present invention, Figure 1 shows a schematic diagram of the recognition order of liver cancer-specific biomarkers.

[0037] In one example, the liver cancer can be hepatocellular carcinoma (HCC), and can be early-stage hepatocellular carcinoma or advanced hepatocellular carcinoma.

[0038] In one example, the expression of the biomarker gene of the present invention can increase specifically for liver cancer.

[0039] In one aspect, the present invention relates to a composition for diagnosing liver cancer, which comprises a preparation for measuring the expression level of one or more biomarker genes selected from the group consisting of alpha-fetoprotein, hyaluronan-mediated motility receptor, neurexophilin 4, paired-like homeodomain 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T at the mRNA or protein level.

[0040] In one example, the above composition may include a preparation for measuring the expression levels of one or more biomarker gene sets selected from the group consisting of alpha-fetoprotein and hyaluronan-mediated motility receptor, alpha-fetoprotein and neuropilin-4, alpha-fetoprotein and paired box 1, alpha-fetoprotein and thrombospondin-4, alpha-fetoprotein and ubiquitin-conjugating enzyme E2T, hyaluronan-mediated motility receptor and neuropilin-4, hyaluronan-mediated motility receptor and paired box 1, hyaluronan-mediated motility receptor and thrombospondin-4, hyaluronan-mediated motility receptor and ubiquitin-conjugating enzyme E2T, neuropilin-4 and paired box 1, neuropilin-4 and thrombospondin-4, neuropilin-4 and ubiquitin-conjugating enzyme E2T, paired box 1 and thrombospondin-4, paired box 1 and ubiquitin-conjugating enzyme E2T, thrombospondin-4 and ubiquitin-conjugating enzyme E2T at the mRNA or protein level.

[0041] In one example, the above composition may include a preparation for measuring the expression levels of one or more biomarker gene sets selected from the group consisting of alpha-fetoprotein, hyaluronan-mediated motility receptor and neuropilin-4; alpha-fetoprotein, hyaluronan-mediated motility receptor and paired box 1; alpha-fetoprotein, hyaluronan-mediated motility receptor and thrombospondin-4; alpha-fetoprotein, hyaluronan-mediated motility receptor and ubiquitin-conjugating enzyme E2T; alpha-fetoprotein, neuropilin-4 and paired box 1; alpha-fetoprotein, neuropilin-4 and thrombospondin-4; alpha-fetoprotein, neuropilin-4 and ubiquitin-conjugating enzyme E2T; alpha-fetoprotein, paired box 1 and thrombospondin-4; alpha-fetoprotein, paired box 1 and ubiquitin-conjugating enzyme E2T; alpha-fetoprotein, thrombospondin-4 and ubiquitin-conjugating enzyme E2T; hyaluronan-mediated motility receptor, neuropilin-4 and paired box 1; hyaluronan-mediated motility receptor, neuropilin-4 and; hyaluronan-mediated motility receptor, neuropilin-4 and thrombospondin-4; hyaluronan-mediated motility receptor, neuropilin-4 and ubiquitin-conjugating enzyme E2T; hyaluronan-mediated motility receptor, paired box 1 and thrombospondin-4; hyaluronan-mediated motility receptor, paired box 1 and ubiquitin-conjugating enzyme E2T; hyaluronan-mediated motility receptor, thrombospondin-4 and ubiquitin-conjugating enzyme E2T; neuropilin-4, paired box 1 and thrombospondin-4; neuropilin-4, paired box 1 and ubiquitin-conjugating enzyme E2T; and neuropilin-4, thrombospondin-4 and ubiquitin-conjugating enzyme E2T at the mRNA or protein level.

[0042] In one example, the preparation for measuring the expression level of the above biomarker gene at the mRNA level may include the nucleic acid sequence of the biomarker, the nucleic acid sequence complementary to the above nucleic acid sequence, a primer pair that specifically recognizes the above nucleic acid sequence and the fragments of the complementary sequence, a probe, or a primer pair and a probe. The above measurement can be carried out by a method selected from the group consisting of polymerase chain reaction, real-time fluorescence quantitative reverse transcription polymerase chain reaction (Real-time RT-PCR), reverse transcription polymerase chain reaction, competitive polymerase chain reaction (Competitive RT-PCR), nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, RNA blot, or DNA chip.

[0043] In one example, the preparation for measuring the expression level of the biomarker gene at the protein level may include an antibody, an antibody fragment, an aptamer, an avidity multimer, or peptidomimetics that specifically recognize the full length of the protein of the above biomarker or its fragment. The above measurement can be carried out by a method selected from the group consisting of immunoblotting, enzyme linked immunosorbent assay, radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorting (FACS), mass spectrometry, or protein microarray.

[0044] The terms "detect" or "measure" used in the present invention refer to quantifying the concentration of the object to be detected or measured.

[0045] In the present invention, the term "primer" refers to a short nucleic acid sequence that has a nucleic acid sequence with a short free 3 hydroxyl group and can form base pairs with a complementary template, and serves as a starting point for the replication of the template strand. The primer can induce DNA synthesis in the presence of reagents for polymerization reaction (i.e., DN polymerase or reverse transcriptase) and different 4 nucleoside triphosphates in an appropriate buffer and temperature.

[0046] In the present invention, the term "probe" refers to a nucleic acid fragment corresponding to several bases to several hundred bases that can specifically bind to mRNA, such as RNA or DNA, etc. Since it is labeled, the presence or absence of a specific mRNA can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. In the present invention, hybridization is carried out using a probe complementary to the above-mentioned alpha-fetoprotein, hyaluronan-mediated motility receptor, neurophilin 4, paired homeodomain 1, thrombospondin 4, and / or ubiquitin-conjugating enzyme E2T gene, and the expression level of the above genes can be diagnosed based on whether hybridization occurs. The selection of a suitable probe and hybridization conditions can be changed based on techniques well known in the art, and there are no particular limitations in the present invention.

[0047] The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. Such nucleic acid sequences can be modified using various means well known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, for example, modification into an uncharged linker (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or a charged linker (e.g., phosphorothioate, dithiophosphonate, etc.).

[0048] In the present invention, suitable conditions for hybridizing a probe with a cDNA molecule can be determined through an optimization step in a series of processes. This step is carried out by those of ordinary skill in the art through a series of processes to establish a protocol for use in a research laboratory. For example, conditions such as temperature, component concentration, hybridization and washing time, buffer components and their pH, and ionic strength depend on various factors such as the length of the probe, the amount of GC, and the target nucleotide sequence. Detailed conditions for hybridization can be confirmed from "Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999)". For example, high stringency conditions in the above stringent conditions refer to hybridizing at 65 °C in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and washing at 68 °C in 0.1× standard saline citrate (SSC) / 0.1% sodium dodecyl sulfate. Alternatively, high stringency conditions refer to washing at 48 °C in 6× standard saline citrate / 0.05% sodium pyrophosphate. Low stringency conditions refer to washing, for example, at 42 °C in 0.2× standard saline citrate / 0.1% sodium dodecyl sulfate.

[0049] In the present invention, the term "antibody" is a term well known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein expressed in the alpha-fetoprotein, hyaluronan-mediated motility receptor, neuropilin 4, paired homeodomain 1, thrombospondin 4, and / or ubiquitin-conjugating enzyme E2T genes, which are used as markers of the present invention. The above antibodies can be prepared using known methods. This includes partial peptides that can be made from the above proteins. There is no particular limitation on the form of the antibody of the present invention. If it is a polyclonal antibody, monoclonal antibody, or any one with antigen-binding properties, a part thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies, such as humanized antibodies.

[0050] In one aspect, the present invention relates to a liver cancer diagnostic kit comprising a composition for diagnosing liver cancer.

[0051] In one example, the above-mentioned kit may further include tools and / or reagents for collecting biological samples from a subject or patient, and tools and / or reagents for preparing genomic DNA, cDNA, RNA or proteins from the samples. For example, it may include PCR primers for amplifying relevant regions of genomic DNA. The above-mentioned kit may include probes for genetic factors that can be used for pharmacogenomic analysis. And when using such a kit, labeled oligonucleotides can be used to easily identify during the analysis process.

[0052] In one example, the above-mentioned kit may further contain labeling substances, such as DNA polymerase, dNTP (dGTP, dCTP, dATP and dTTP), fluorescent substances, etc.

[0053] In the present invention, the term "liver cancer diagnostic kit" refers to a kit containing the composition for diagnosing liver cancer of the present invention. Therefore, the above expression "liver cancer diagnostic kit" can be used interchangeably or mixed with "composition for diagnosing liver cancer". In this specification, the term "diagnosis" includes determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, determining the prognosis of an object suffering from a specific disease or disorder (for example, identifying pre-metastatic or metastatic cancer symptoms, determining the stage of cancer or the responsiveness of cancer to treatment), or therametrics (for example, monitoring the status of an object to provide information related to treatment efficacy).

[0054] In the present invention, the term "diagnostic biomarker, biomarker for diagnosis or diagnosis marker" is a substance capable of diagnosing the presence of liver cancer cells or tissues separately from normal cells or tissues, including organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that are in an increased or decreased expression state in cells or tissues having liver cancer cells as compared with normal cells. For the purposes of the present invention, the above-mentioned liver cancer detection or diagnosis biomarker is one or more selected from the group consisting of gene alpha-fetoprotein, hyaluronan-mediated motility receptor, neuropilin 4, paired homeodomain 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T, and is a gene with specifically increased mRNA expression or protein expression level in liver cancer.These markers include not only genes, but also DNA or mRNA complementary to a marker, and are preferably composite markers including two or more of these markers, more preferably selected from the group consisting of alpha-fetoprotein and hyaluronan-mediated motility receptor; alpha-fetoprotein and neuropilin-4; alpha-fetoprotein and paired homeodomain 1; alpha-fetoprotein and thrombospondin-4; alpha-fetoprotein and ubiquitin-conjugating enzyme E2T; hyaluronan-mediated motility receptor and neuropilin-4; hyaluronan-mediated motility receptor and paired homeodomain 1; hyaluronan-mediated motility receptor and thrombospondin-4; hyaluronan-mediated motility receptor and ubiquitin-conjugating enzyme E2T; neuropilin-4 and paired homeodomain 1; neuropilin-4 and thrombospondin-4; neuropilin-4 and ubiquitin-conjugating enzyme E2T; paired homeodomain 1 and thrombospondin-4; paired homeodomain 1 and ubiquitin-conjugating enzyme E2T; thrombospondin-4 and ubiquitin-conjugating enzyme E2T; alpha-fetoprotein, hyaluronan-mediated motility receptor and neuropilin-4; alpha-fetoprotein, hyaluronan-mediated motility receptor and paired homeodomain 1; alpha-fetoprotein, hyaluronan-mediated motility receptor and thrombospondin-4; alpha-fetoprotein, hyaluronan-mediated motility receptor and ubiquitin-conjugating enzyme E2T; alpha-fetoprotein, neuropilin-4 and paired homeodomain 1; alpha-fetoprotein, neuropilin-4 and thrombospondin-4; alpha-fetoprotein, neuropilin-4 and ubiquitin-conjugating enzyme E2T; alpha-fetoprotein, paired homeodomain 1 and thrombospondin-4; alpha-fetoprotein, paired homeodomain 1 and ubiquitin-conjugating enzyme E2T; alpha-fetoprotein, thrombospondin-4 and ubiquitin-conjugating enzyme E2T; hyaluronan-mediated motility receptor, neuropilin-4 and paired homeodomain 1; hyaluronan-mediated motility receptor, neuropilin-4 and thrombospondin-4; hyaluronan-mediated motility receptor, neuropilin-4 and ubiquitin-conjugating enzyme E2T; hyaluronan-mediated motility receptor, paired homeodomain 1 and thrombospondin-4; hyaluronan-mediated motility receptor, paired homeodomain 1 and ubiquitin-conjugating enzyme E2T; hyaluronan-mediated motility receptor, thrombospondin-4 and ubiquitin-conjugating enzyme E2T; neuropilin-4, paired homeodomain 1 and thrombospondin-4; neuropilin-4, paired homeodomain 1 and ubiquitin-conjugating enzyme E2T; and neuropilin-4, thrombospondin-4 and ubiquitin-conjugating enzyme E2T, and one or more selected from the group consisting of these combinations.

[0055] In one aspect, the present invention relates to a method for screening anti-cancer candidate substances, which comprises: step (a), measuring the expression levels of alpha-fetoprotein, hyaluronan-mediated motility receptor, neuropilin 4, paired-like homeodomain 1, thrombospondin 4 or ubiquitin-conjugating enzyme E2T genes in liver cancer cells; step (b), administering an anti-cancer candidate substance to the above liver cancer cells and measuring the expression levels of alpha-fetoprotein, hyaluronan-mediated motility receptor, neuropilin 4, paired-like homeodomain 1, thrombospondin 4 or ubiquitin-conjugating enzyme E2T genes; and step (c), when the expression levels of alpha-fetoprotein, hyaluronan-mediated motility receptor, neuropilin 4, paired-like homeodomain 1, thrombospondin 4 or ubiquitin-conjugating enzyme E2T genes in step (a) are greater than the expression levels of alpha-fetoprotein, hyaluronan-mediated motility receptor, neuropilin 4, paired-like homeodomain 1, thrombospondin 4 or ubiquitin-conjugating enzyme E2T genes in step (b), determining that the above anti-cancer candidate substance is an effective anti-cancer substance.

[0056] In one aspect, the present invention relates to a method for providing information required for diagnosing liver cancer, which comprises: step (a), measuring the expression levels of one or more biomarker genes selected from the group consisting of alpha-fetoprotein, hyaluronan-mediated motility receptor, neuropilin 4, paired-like homeodomain 1, thrombospondin 4 and ubiquitin-conjugating enzyme E2T in a biological sample isolated from a subject; step (b), comparing with the corresponding results of the corresponding biomarkers in a normal control group sample; and step (c), when the expression level of the biomarker gene in step (a) is greater than the expression level of the biomarker gene in step (b), determining that the above subject may have liver cancer.

[0057] In one example, the above method may further comprise a step of distinguishing early-stage liver cancer and advanced liver cancer according to the expression change level of the biomarker gene.

[0058] In one example, the specific method for measuring the expression level of the above biomarker gene at the mRNA or its protein level is as follows: the expression of the above gene can be detected at the mRNA level or protein level, mRNA or protein can be isolated from a biological sample using known procedures, and the expression level of the gene can be confirmed by reverse transcriptase-polymerase chain reaction or real-time polymerase chain reaction.

[0059] In one example, the biological sample may include samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, etc., and more preferably whole blood, serum or plasma.

[0060] The present invention will be described in more detail by the following embodiments. However, the following embodiments are only used to specifically describe the content of the present invention, and the present invention is not limited thereto. Detailed implementation manners

[0061] Example 1. Blood biomarker screening using a database

[0062] 1 - 1. Screening of Hepatocellular Carcinoma Cell - Specific Markers

[0063] Three independent groups were selected from blood samples (15 normal liver patient samples (Normal liver, NL); 20 chronic hepatitis patient samples (Chronic hepatitis, CH); 10 liver cirrhosis patient samples (Liver Cirrhosis, LC); 18 early hepatocellular carcinoma patient samples (Early HCC, eHCC); and 45 advanced hepatocellular carcinoma patient samples (Advanced HCC)) of an independent cohort of liver disease patients (108 samples from 86 patients). After extracting total RNA using TRIzol reagent, a sequencing library was prepared using the RNA Library Prep Kit for Illumina (Cat#E7420L), and sequencing was performed according to the standard method of Illumina by Illumina HiSeq 2000. After mapping the entire transcriptome of the analyzed liver using STAR and Gencode v.25, the expression profile was replaced with FPKM values, then the gene types were classified using Gencode v.25, and 12,654 signal peptides were derived using SignalP 4.1. All the generated data were recorded in the open Omix database GEO. After that, 2,502 pre-cancer genomes overexpressed only in hepatocellular carcinoma were derived ( Figure 2 ), and analysis was performed through the Cancer Genome Atlas hepatocellular carcinoma (TCGA_LIHC) data and the Gene Expression Omnibus (GEO) database, and 737 genes overexpressed in both of these two databases were analyzed by hierarchical clustering. As a result, the GSE114564 database showed a phylogenetic tree divided into 5 subclasses: normal liver, chronic hepatitis (CHB), liver cirrhosis, early hepatocellular carcinoma, and advanced hepatocellular carcinoma, and the TCGA_LIHC database showed a phylogenetic tree divided into 2 subclasses: normal liver and advanced hepatocellular carcinoma ( Figure 3)。When comparing the expression status of the 737 genes calculated in two datasets, it was confirmed that there were significant differences in the expression changes in the progression to liver cancer or in advanced liver cancer compared with normal liver tissue. And, in order to analyze the gene data with two categories, gene set enrichment analysis (GSEA) was used for the analysis. The above gene set enrichment analysis is used to extract important gene sets in which the expression values of the two categories show significant differences statistically in various gene sets composed based on biological characteristics. From the analysis results, it was confirmed that there was a very close association with the dataset of a well-known liver cancer cohort gene set CHANG_LIVER_CANCER in the past (mean agglutination index NES = 1.88, NES = 1.85). Then, by identifying 10 candidate marker genes ([ Figures 4a - 4d ) that were overexpressed in both the above GSE114564 data cohort and GSE6764, 10 candidate markers CCNB2, CDT1, COCH, CSMD1, HMMR, NXPH4, OLFML2B, PITX1, THBS4, and UBE2T with increased expression specificity only in hepatocellular carcinoma were screened out.

[0064] 1 - 2. Verification of the Difference between Normal Liver and Progressive Hepatocellular Carcinoma Using TCGA_LIHC and ICGC_LIRI

[0065] In order to achieve a biomarker that is significantly increased in the serum of patients, the increase rate of expression in advanced liver cancer must be significantly higher than that of normal liver statistically. In the publicly available data, based on sequencing that can accurately measure expression, and separately analyzing and verifying the expression levels of 10 marker genes in non-tumor tissues and tumor tissues of liver cancer patients from the TCGA_LIHC dataset and ICGC_LIRI dataset as large-scale cohorts, it was confirmed that there were obvious expression differences in both cohorts ( Figures 5a - 5d ).

[0066] 1 - 3. Analysis of 50 Matched Pairs of Hepatocellular Carcinoma Patients

[0067] The GSE77314 dataset was used to conduct a comparative analysis of the expression levels of the above 10 marker genes. The above GSE77314 dataset is the expression values of genes measured by sequencing methods in the surrounding normal tissues and liver cancer tissues of 50 liver cancer patients as a Chinese liver cancer patient cohort. As a result, in most patients, it was confirmed that the expression was significantly increased in liver cancer tissues compared with normal liver tissues ( Figures 6a - 6b ).

[0068] Example 2. Enzyme-linked immunosorbent assay analysis of the first selected markers

[0069] 2 - 1. Confirmation of the Expression Profile of Markers

[0070] In a cohort of liver disease patients (771 specimens from 100 patients) with independent blood specimens for alpha-fetoprotein values, which are cancer markers for liver cancer cells (135 specimens from 16 normal liver patients (Normal liver, NL); 65 specimens from 13 patients with chronic hepatitis (Chronic hepatitis, CH); 103 specimens from 15 patients with liver cirrhosis (Liver Cirrhosis, LC); 227 specimens from 35 patients with early hepatocellular carcinoma (Early HCC, eHCC); and 241 specimens from 24 patients with advanced hepatocellular carcinoma (Advanced HCC, avHCC))( Figure 7 ), the expression levels of the 10 marker genes selected in Example 1 above were confirmed by enzyme-linked immunosorbent assay analysis( Figures 8a - 8b ).

[0071] As a result, in the case of CCNB2, the average value measured from normal liver patients (NL) was 0.02 ng / ml, chronic hepatitis patients (CH) showed 0.2029 ng / ml, liver cirrhosis patients (LC) showed 0.43 ng / ml, early hepatocellular carcinoma patients (eHCC) showed 0.27 ng / ml, and advanced hepatocellular carcinoma patients (avHCC) showed 0.31 ng / ml. Thus, LC showed the maximum value. In the case of CDT1, the average value measured from normal liver patients (NL) was 167.7 pg / ml, CH showed 230.8 pg / ml, LC showed 178.2 pg / ml, eHCC showed 103.5 pg / ml, and avHCC showed 146.8 pg / ml. Thus, avHCC showed the maximum value. Generally, no significant differences were shown among the diseases. In the case of COCH, the average value measured from normal liver patients (NL) was 1.724 ng / ml, CH showed 12.78 ng / ml, LC showed 10.03 ng / ml, eHCC showed 6.74 ng / ml, and avHCC showed 8.025 ng / ml. Therefore, except for normal liver, relatively high values were shown at all stages of liver diseases and liver cancer. In the case of CSMD1, the average value measured from normal liver patients (NL) was 14.8 ng / ml, CH showed 11.65 ng / ml, LC showed 14.48 ng / ml, eHCC showed 14.72 ng / ml, and avHCC showed 15.66 ng / ml. Thus, generally similar values were shown. In the case of OLFML2B, NL showed an average of 175.2 pg / ml, CH showed 658.8 pg / ml, LC showed 338.4 pg / ml, eHCC showed 284.1 pg / ml, and avHCC showed 349.6 pg / ml. Therefore, except for normal liver, relatively high values were shown at all stages of liver diseases and liver cancer, especially relatively high in CH. In the case of HMMR, the average value measured from NL was 0.21 ng / ml, CH showed 0.62 ng / ml, LC showed 0.74 ng / ml, eHCC showed 1.54 ng / ml, and avHCC showed 1.64 ng / ml. Thus, similar to the sequencing results, this value increased with the progression of the liver disease stage. In the case of NXPH4, NL showed an average of 3.54 ng / ml, CH showed 10.23 ng / ml, LC showed 6.52 ng / ml, eHCC showed 15.02 ng / ml, and avHCC showed 19.83 ng / ml. Thus, it was slightly higher in CH, but similar to the sequencing results, this value increased with the progression of the liver disease stage.In the case of PITX1, NL showed an average of 2042 pg / ml, CH showed 1994 pg / ml, LC showed 3238 pg / ml, eHCC showed 3314 pg / ml, and avHCC showed 6135 pg / ml. Thus, a value lower than the normal value was shown in CH, but similar to the sequencing results, this value increased as the liver disease stage progressed. In the case of THBS4, NL showed an average of 45.36 ng / ml, CH showed 70.96 ng / ml, LC showed 141.8 ng / ml, eHCC showed 229.4 ng / ml, and avHCC showed 233.6 ng / ml. Thus, the same as the sequencing results, this value increased as the liver disease stage progressed. In the case of UBE2T, NL showed an average of 16.14 ng / ml, CH showed 319.9 ng / ml, LC showed 426.1 ng / ml, eHCC showed 505.5 ng / ml, and avHCC showed 877.2 ng / ml. Thus, it increased approximately 20-fold in liver diseases compared to normal liver, and this value increased as the liver disease stage progressed.

[0072] 2 - 2. Receiver Operating Characteristic Curve Analysis

[0073] The receiver operating characteristic curve analysis was performed on 10 marker genes in the above cohort by ELISA values.

[0074] As a result, CSMD1, HMMR, NXPH4, OPITX1, THBS4, and UBE2T had statistically significant values compared to the reference line, and showed an area under the curve (AUC) value similar to or greater than that of alpha-fetoprotein in the receiver operating characteristic curve analysis, indicating that the markers with specificity and sensitivity were HMMR, NXPH4, PITX1, THBS4, and UBE2T( Figures 9a - 9b ).

[0075] Example 3. Verification of Early Cancer Diagnosis Markers HMMR, NXPH4, PITX1, THBS4, and UBE2T

[0076] 3 - 1. Confirmation of the Expression Profile of Markers

[0077] In a cohort of liver disease patients (1148 samples from 279 patients) with independent AFP values as a cancer biomarker for liver cancer cells, blood samples were used (222 samples from 49 normal liver patients (Normal liver, NL); 115 samples from 31 chronic hepatitis patients (Chronic hepatitis, CH); 183 samples from 46 liver cirrhosis patients (Liver Cirrhosis, LC); 345 samples from 77 early hepatocellular carcinoma patients (Early HCC, eHCC); and 283 samples from 64 advanced hepatocellular carcinoma patients (Advanced HCC, avHCC))( Figure 10 ), and the expression levels of the 10 biomarker genes selected in Example 1 above were confirmed by enzyme-linked immunosorbent assay analysis( Figures 11a - 11b ).

[0078] As a result, when measuring the protein expression levels of 5 biomarkers separately in the validation cohort, in the case of HMMR, comparing the normal group with each liver disease group in a total of 230 samples showed very significant differences except for the cirrhosis group, and it was confirmed that its expression was specifically high especially in early liver cancer. In the case of NXPH4, compared with the normal group, each liver disease stage group showed significant expression changes, and the same result was shown for PITX1. In the case of THBS4, like HMMR, its expression increased significantly except for the cirrhosis group, and a relatively large value was shown in the early liver cancer group. In the case of UBE2T, like the test cohort, it was not expressed at all in the normal group, but showed increased expression in the liver disease stage groups.

[0079] 3 - 2. Receiver Operating Characteristic Curve Analysis

[0080] Receiver operating characteristic curve analysis was performed on the biomarker genes HMMR, NXPH4, PITX1, THBS4, and UBE2T in the above cohort.

[0081] As a result, in the cases of HMMR and THBS4, values of AUC = 0.856 and AUC = 0.772 were shown respectively, and thus it was confirmed that their expression levels were higher than the AUC = 0.749 of the previous biomarker AFP( Figures 12a - 12b ).

[0082] 3 - 3. Confirmation of the Expression Status at Each Development Stage of Hepatocellular Carcinoma

[0083] To confirm the sensitivity, specificity, and accuracy of the above-mentioned HMMR, NXPH4, PITX1, THBS4, and UBE2T that passed AFP verification, an enzyme-linked immunosorbent assay analysis was performed on the above-mentioned cohort specimens.

[0084] Table 1

[0085]

[0086] The results are shown in the above Table 1 and Figures 13a - 13g. Specifically, the following steps were carried out for alpha-fetoprotein and five markers: 1) comparison was made between non-hepatocellular carcinoma specimens (normal liver, hepatitis, cirrhosis specimens) and hepatocellular carcinoma specimens; 2) after comparison between liver disease specimens (hepatitis, cirrhosis specimens) and hepatocellular carcinoma specimens, only for early-stage hepatocellular carcinoma, analysis was specifically carried out according to 3) non-hepatocellular carcinoma specimens and 4) liver disease specimens. In all four cases, the sensitivity, specificity, and accuracy of the hyaluronan-mediated motility receptor were found to be the greatest. When the cut-off values of their respective markers were determined using the MedCal program, the hyaluronan-mediated motility receptor was measured to be 0.8 ng / μl, neurophilin 4 was 7.5 ng / μl, paired-like homeodomain 1 was 2475 pg / μl, thrombospondin 4 was 90 ng / μl, and ubiquitin-conjugating enzyme E2T was 40 ng / μl. In the case of specimens with cut-off values increased beyond each cut-off value, analysis was carried out by classifying them as positive or low negative. From the positive rates in normal liver, the alpha-fetoprotein was measured to be 2%, the hyaluronan-mediated motility receptor was 0%, neurophilin 4 was 6%, paired-like homeodomain 1 was 23%, thrombospondin 4 was 4%, and ubiquitin-conjugating enzyme E2T was 0%. In the hepatitis group, the alpha-fetoprotein was measured to be 19%, the hyaluronan-mediated motility receptor was 19%, neurophilin 4 was 50%, paired-like homeodomain 1 was 44%, thrombospondin 4 was 44%, and ubiquitin-conjugating enzyme E2T was 63%. In the cirrhosis group, the alpha-fetoprotein was measured to be 39%, the hyaluronan-mediated motility receptor was 17%, neurophilin 4 was 48%, paired-like homeodomain 1 was 57%, thrombospondin 4 was 4%, and ubiquitin-conjugating enzyme E2T was 70%. In the early-stage hepatocellular carcinoma group, the alpha-fetoprotein was measured to be 33%, the hyaluronan-mediated motility receptor was 83%, neurophilin 4 was 64%, paired-like homeodomain 1 was 72%, thrombospondin 4 was 54%, and ubiquitin-conjugating enzyme E2T was 54%. Thus, the positive rates of the five markers measured were significantly higher than those of alpha-fetoprotein as a marker for hepatocellular carcinoma measurement. In the advanced hepatocellular carcinoma group, the alpha-fetoprotein was shown to be 73%, the hyaluronan-mediated motility receptor was 78%, neurophilin 4 was 87%, paired-like homeodomain 1 was 89%, thrombospondin 4 was 62%, and ubiquitin-conjugating enzyme E2T was 67%. Then, when comparing the positive rates of alpha-fetoprotein and the five markers in hepatocellular carcinoma patients, the alpha-fetoprotein was 52%, while the remaining markers showed high positive rates. Especially when comparing the positive rates of each marker in hepatocellular carcinoma patients with negative alpha-fetoprotein, in the case of HMMR, a high positive rate of 86% was measured, which is expected to be able to supplement hepatocellular carcinoma patients with undetected positive alpha-fetoprotein. In the case of the early-stage hepatocellular carcinoma group, the alpha-fetoprotein showed a positive rate of 33%. On the contrary, in the case of HMMR, a high positive rate of 83% was shown.Moreover, in liver cancer patients with negative alpha-fetoprotein, a high positive rate of 85% was also shown.

[0087] Example 4. Confirmation of the Combined Effect of Early Cancer Diagnosis Markers

[0088] For the cohort of Example 3-1 above, the diagnostic efficacy of hepatocellular carcinoma was compared according to combinations of two markers among alpha-fetoprotein, hyaluronan-mediated motility receptor, neuropilin-4, paired box 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T (combinations of alpha-fetoprotein with hyaluronan-mediated motility receptor, neuropilin-4, paired box 1, thrombospondin 4, or ubiquitin-conjugating enzyme E2T; or combinations of two markers among hyaluronan-mediated motility receptor, neuropilin-4, paired box 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T) or combinations of three markers (combinations of alpha-fetoprotein with two markers among hyaluronan-mediated motility receptor, neuropilin-4, paired box 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T; or combinations of three markers among hyaluronan-mediated motility receptor, neuropilin-4, paired box 1, thrombospondin 4, and ubiquitin-conjugating enzyme E2T).

[0089] Table 2

[0090]

[0091] The results are shown in Table 2 above and Figures 14a - 14d. Specifically, when combining two markers, if all liver cancer patients are used as subjects, the combination of alpha-fetoprotein and hyaluronan-mediated motility receptor shows a positive rate of 92%, and the combination of hyaluronan-mediated motility receptor and paired homeobox 1 shows a maximum positive rate of 96%. If early-stage liver cancer patients are used as subjects, the combination of alpha-fetoprotein and hyaluronan-mediated motility receptor shows a positive rate of 90%, and the combination of hyaluronan-mediated motility receptor and paired homeobox 1 shows a maximum positive rate of 99%. Moreover, when combining three markers, if all liver cancer patients are used as subjects, the combinations of alpha-fetoprotein, hyaluronan-mediated motility receptor and paired homeobox 1, hyaluronan-mediated motility receptor, neuropilin 4 and paired homeobox 1, and hyaluronan-mediated motility receptor, paired homeobox 1 and ubiquitin-conjugating enzyme E2T show a positive rate of 100%. If early-stage liver cancer patients are used as subjects, the combinations of alpha-fetoprotein and hyaluronan-mediated motility receptor and paired homeobox 1, hyaluronan-mediated motility receptor, neuropilin 4 and paired homeobox 1, hyaluronan-mediated motility receptor, neuropilin 4 and ubiquitin-conjugating enzyme E2T, and hyaluronan-mediated motility receptor, paired homeobox 1 and ubiquitin-conjugating enzyme E2T show a positive rate of 100%.

[0092] Furthermore, when performing a receiver operating characteristic analysis on the combinations that showed a 100% positive rate in 86 non-hepatocellular carcinoma specimens and 132 hepatocellular carcinoma specimens, it was confirmed that, compared with the previous alpha-fetoprotein, the area under the curve values of all combinations were significantly increased statistically. Among the two combinations, the combination of alpha-fetoprotein and hyaluronan-mediated motility receptor was evaluated as the best. Among the three combinations, the combination of alpha-fetoprotein, hyaluronan-mediated motility receptor, and paired homeodomain 1 showed the maximum value. In terms of diagnostic analysis, among the two combinations, for the results of the accuracy analysis, the highest values were measured for hyaluronan-mediated motility receptor and paired homeodomain 1, and the odds ratio also showed the highest value of 75.23. Among the three combinations, for the results of the accuracy analysis, alpha-fetoprotein, hyaluronan-mediated motility receptor, and paired homeodomain 1 showed the highest value of 90.37%, and the odds ratio also showed the highest value of 87.04. Also, when performing a receiver operating characteristic analysis on the combinations that showed a 100% positive rate in 86 non-hepatocellular carcinoma specimens and 69 early hepatocellular carcinoma specimens respectively, it was confirmed that, compared with the previous alpha-fetoprotein, the area under the curve values of all combinations were significantly increased statistically. Among the two combinations, the combination of hyaluronan-mediated motility receptor and paired homeodomain 1 was evaluated as the best. Among the three combinations, the combination of alpha-fetoprotein, hyaluronan-mediated motility receptor, and paired homeodomain 1 showed the maximum value. In terms of diagnostic analysis, among the two combinations, for the results of the accuracy analysis, the highest value of 88.39% was measured for hyaluronan-mediated motility receptor and paired homeodomain 1, and the odds ratio also showed the highest value of 64.75. Among the three combinations, for the results of the accuracy analysis, alpha-fetoprotein, hyaluronan-mediated motility receptor, and paired homeodomain 1 showed the highest value of 92.75%, and the odds ratio was also measured as the highest value of 65.83.

Claims

1. A biomarker for diagnosing hepatocellular carcinoma, characterized in that, it comprises the genes of alpha-fetoprotein, hyaluronan-mediated motility receptor and paired-like homeodomain 1, or the proteins expressed from the above genes.

2. The biomarker for diagnosing hepatocellular carcinoma according to claim 1, characterized in that, the hepatocellular carcinoma is early-stage hepatocellular carcinoma or advanced hepatocellular carcinoma.

3. A composition for diagnosing hepatocellular carcinoma, characterized in that, it comprises a preparation for measuring the expression levels of the biomarker genes of alpha-fetoprotein, hyaluronan-mediated motility receptor and paired-like homeodomain 1 at the mRNA or protein level.

4. The composition for diagnosing hepatocellular carcinoma according to claim 3, characterized in that, the hepatocellular carcinoma is early-stage hepatocellular carcinoma or advanced hepatocellular carcinoma.

5. The composition for diagnosing hepatocellular carcinoma according to claim 3, characterized in that, the preparation for measuring the expression level of the biomarker gene at the mRNA level is a primer pair, a probe or a primer pair and a probe that specifically recognize the above gene.

6. The composition for diagnosing hepatocellular carcinoma according to claim 5, characterized in that, the measurement is carried out by a method selected from the group consisting of polymerase chain reaction, real-time fluorescence quantitative reverse transcription polymerase chain reaction, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, nuclease protection assay, in situ hybridization method, nucleic acid microarray, RNA blot or DNA chip.

7. The composition for diagnosing hepatocellular carcinoma according to claim 3, characterized in that, the preparation for measuring the expression level of the biomarker gene at the protein level is an antibody, an antibody fragment, an aptamer, a high-affinity multimer or a peptidomimetic that specifically recognizes the full length of the above biomarker protein.

8. The composition for diagnosing hepatocellular carcinoma according to claim 7, characterized in that, the measurement is carried out by a method selected from the group consisting of immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorting, mass spectrometry or protein microarray.

9. The composition for diagnosing hepatocellular carcinoma according to claim 8, characterized in that, the measurement is carried out by radioimmunodiffusion method.

10. A hepatocellular carcinoma diagnostic kit, characterized in that, it comprises the composition for diagnosing hepatocellular carcinoma according to claim 3.

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