A composition for diagnosing liver cancer utilizing changes in the CPG methylation of specific genes, and its use.

Abstract

To provide a composition for diagnosing liver cancer by using CPG methylation changes in specific genes, and use thereof.SOLUTION: The present invention relates to a composition, a kit, a nucleic acid chip and a method which allow diagnosis of liver cancer by detecting methylation levels of CpG sites of any one or more genes selected from a group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36 and VANGL2. The present invention facilitates accurate and rapid diagnosis of liver cancer, and also permits diagnosis at an early stage.SELECTED DRAWING: Figure 14

Description

Technical Field

[0001] The present invention relates to a composition, a kit, a nucleic acid chip, and a method for diagnosing liver cancer by detecting the methylation level of CpG sites of any one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2.

Background Art

[0002] This application claims priority to Korean Patent Application No. 10-2019-0127218, filed on October 14, 2019, the entire specification of which is incorporated herein by reference.

[0003] Liver cancer is a type of cancer with a high incidence rate worldwide. The mortality rate of liver cancer in Korea is 23 per 100,000 population, which is very high. Approximately 10% of the total mortality rate of Koreans is related to hepatitis, cirrhosis, and liver cancer. Since liver cancer has no perceptible symptoms in the early stage of occurrence, early diagnosis is difficult. Usually, most liver cancers are progressive carcinomas that cannot be treated appropriately and are discovered in a progressive state, so treatment is very limited and the prognosis is extremely poor. Since the prognosis of liver cancer varies greatly depending on the progression state of the cancer at the time of diagnosis, early detection of liver cancer patients is very important for increasing the survival rate of liver cancer patients.

[0004] On the other hand, epigenetics is a field that studies the regulation of gene expression that occurs without changes in the DNA base sequence. Epigenetics studies the regulation of gene expression by epigenetic mutations such as DNA methylation, miRNA, or histone acetylation, methylation, phosphorylation, and ubiquitination. Double DNA methylation is the most extensively studied epigenetic mutation. Epigenetic mutations can lead to changes in gene function and transformation into tumor cells. Therefore, DNA methylation is associated with the expression (or suppression and induction) of intracellular disease-regulating genes, and recently, cancer diagnostic methods using DNA methylation measurement have been proposed. In particular, since cancer-specific methylation can occur even in precancerous tissues, the detection of cancer-specific methylation has high potential for use in cancer diagnosis.

[0005] Therefore, there is a need to develop effective liver cancer-specific methylation markers that can predict the risk of liver cancer. [Overview of the project] [Problems that the invention aims to solve]

[0006] Here, the present inventors have discovered that a specific gene CpG site is hypermethylated in liver cancer, and have completed the present invention by developing a composition, kit, nucleic acid chip, and method that can diagnose liver cancer by detecting the methylation level.

[0007] Therefore, the object of the present invention is to provide a liver cancer diagnostic composition comprising a formulation for measuring the methylation level of a specific gene's CpG site.

[0008] Another object of the present invention is to provide a liver cancer diagnostic kit containing a PCR primer pair for amplifying a fragment containing a CpG site of a specific gene and a sequencing primer for pyrosequencing the PCR product amplified by the primer pair.

[0009] Another object of the present invention is to provide a nucleic acid chip for liver cancer diagnosis in which a fragment containing a CpG site of a specific gene and a probe that can be hybridized under harsh conditions are immobilized.

[0010] Another object of the present invention is to provide a method for diagnosing liver cancer, which includes measuring and comparing the methylation levels of CpG sites of specific genes from different samples.

[0011] Another object of the present invention is to provide a formulation for measuring the methylation level of a gene CpG site for the production of a liver cancer diagnostic formulation. [Means for solving the problem]

[0012] To achieve the above objective, the present invention provides a liver cancer diagnostic composition comprising a formulation for measuring the methylation level of a CpG site of one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2.

[0013] To achieve another objective of the present invention, the present invention provides a liver cancer diagnostic kit comprising a primer pair for amplifying a fragment containing a CpG site of any one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2.

[0014] Furthermore, to achieve yet another objective of the present invention, the present invention provides a nucleic acid chip for liver cancer diagnosis on which a probe capable of hybridizing with a fragment containing a CpG site of any one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 is immobilized.

[0015] Furthermore, in order to achieve yet another objective of the present invention, the present invention provides a step of measuring the methylation level of the CpG site of one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36 and VANGL2 from a sample of a patient suspected of having liver cancer; and The present invention provides a method for diagnosing liver cancer, comprising the step of comparing the measured methylation level with the methylation level of the CpG site of the same gene in a normal control sample.

[0016] Furthermore, to achieve yet another objective, the present invention provides the use of a formulation for measuring the methylation level of a CpG site of any one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 for the production of a hepatocellular carcinoma diagnostic formulation.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The following references provide one of the skills that have general definitions of several terms used in the specification of this invention: Singleton et al. DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOTY (2nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND D TECHNOLOGY (Walkered., 1988); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY.

[0018] The present invention will be described in detail below.

[0019] The present invention provides a liver cancer diagnostic composition comprising a drug that measures the methylation level of a CpG site of one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2.

[0020] In this invention, "methylation" means the attachment of a methyl group to a base that makes up DNA. Preferably, the presence or absence of methylation in this invention refers to the presence or absence of methylation occurring at the cytosin of a specific CpG site of a specific gene. When methylation occurs, the binding of transcription factors is inhibited, and the expression of a specific gene is suppressed. Conversely, when demethylation or hypomethylation occurs, the expression of a specific gene increases. In addition to A, C, G, and T, mammalian cell genomic DNA contains a fifth base called 5-methylcytosine (5-methylcytosine, 5-mC), which has a methyl group attached to the fifth carbon of the cytosine ring. Methylation of 5-methylcytosine occurs only at the carbon of a CG dinucleotide (5'-mCG-3') called CpG, and methylation of CpG suppresses the expression of alu or transposons and genomic repeat sequences. Furthermore, since the 5-mC of CpG is readily deaminated to thymine (T), CpG is a site where most genetic changes frequently occur in mammalian cells.

[0021] In the present invention, the term "measurement of methylation level" refers to measuring the methylation level of the CpG site of one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2, and can be measured by a bisulfite-treated detection method or a bisulfite-independent detection method. Methylation levels can be measured by methylation-specific PCR, such as methylation-specific polymerase chain reaction (MSP), real-time methylation-specific polymerase chain reaction, PCR using methylated DNA-specific binding proteins, or quantitative PCR. It can also be measured by automated base analysis such as pyrosequencing and bisulfite sequencing, but is not limited to these methods. Furthermore, as a bisulfite-independent detection method, it can be measured using a detection method employing TET protein (ten-eleven translocation protein) (see Nature Biotechnology, volume 37, pages 424-429 (2019)). The aforementioned TET protein is an enzyme that acts on DNA and is involved in the chemical change of bases. Unlike bisulfite treatment, where all C bases except methylated C are converted to T bases, Tet protein converts only methylated C to T, enabling more efficient detection.

[0022] Preferably, the CpG site of any one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36 and VANGL2 means the CpG site present on the DNA of the gene. The DNA of the gene is a concept that includes all a series of structural units necessary for the gene to be expressed and are operably linked to each other. For example, it includes a promoter region, a protein coding region (open reading frame, ORF) and a terminator region. Thus, FAM The CpG site of any one or more genes selected from the group consisting of 110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36 and VANGL2 may be present in the promoter region, protein coding region or terminator region of the corresponding gene, etc. <000,0083><000,0084>Preferably, in the present invention, measuring the methylation level of the CpG site of any one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36 and VANGL2 may mean measuring the methylation level of cytosine at the CpG site of the gene described in Table 1 below.

[0024] [Table 1]

[0025] In the present invention, the CpG site of any one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36 and VANGL2 is characterized in that it is located between + / −2000 bases (2 kb) from the transcription start site (transport start site, TSS) of the gene.

[0026] In the present invention, the nucleotide sequence of the human genomic chromosomal region was expressed according to the February 2009 Human reference sequence (GRCh37). However, the specific sequence of the human genomic chromosomal region may be slightly changed as the research results of the genomic sequence are updated. Due to such changes, the expression of the human genomic chromosomal region of the present invention may also be different. Therefore, even if the human reference sequence is updated after the filing date of the present invention and the expression of the human genomic chromosomal region is changed to be different from the current one, it is obvious that the scope of the present invention extends to the changed human genomic chromosomal region. Such change contents can be easily known by anyone with ordinary knowledge in the technical field to which the present invention belongs. It is a matter that can be known.

[0027] In the present invention, the agent for measuring the methylation level of the CpG site may include a compound that modifies cytosine bases or a methylation-sensitive restriction enzyme, primers specific to the sequence of the methylated allele of any one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36 and VANGL2, and primers specific to the sequence of the unmethylated allele.

[0028] The compound that deforms the cytosine base is a compound that deforms unmethylated cytosin or methylated cytosin, and may be, but not limited to, a bisulfite or salt thereof that deforms unmethylated cytosin, preferably sodium bisulfite, or a TET protein that deforms methylated cytosin. Methods for detecting whether or not the CpG site is methylated by deforming such a cytosine base are widely known in the industry (WO01 / 26536;US2003 / 0148326A1). Furthermore, the methylation-sensitive restriction enzyme is a restriction enzyme that can specifically detect methylation of the CpG site, and may be a restriction enzyme that contains CG as the recognition site of the restriction enzyme. Examples include, but are not limited to, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, and NotI. Whether or not cleavage by the restriction enzyme occurs differs depending on whether or not the C at the restriction enzyme recognition site is methylated or unmethylated, and this can be detected by PCR or Southern blot analysis. Other methylation-sensitive restriction enzymes other than the restriction enzymes described above are well known in the industry. The primers may include primers specific to the methylated allele sequence and primers specific to the unmethylated allele sequence of one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2. In this invention, the term "primer" means a short nucleic acid sequence having a short free 3' terminal hydroxyl group that can form base pairs with a complementary template and function as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in a suitable buffer solution and temperature. The primer may also incorporate additional features that do not alter the basic properties of the primer acting as a starting point for DNA synthesis, such as sense and antisense nucleic acids having 7 to 50 nucleotide sequences. The primers of the present invention can preferably be designed according to the sequence of a specific CpG site to be analyzed for the presence or absence of methylation, and more preferably, one or more selected from the group consisting of a primer pair that can specifically amplify cytosin that is methylated and not deformed by bisulfite, a primer pair that can specifically amplify cytosin that is not methylated and deformed by bisulfite, a primer pair that can specifically amplify cytosin that is methylated and deformed by Tet-system proteins, and a primer pair that can specifically amplify cytosin that is not methylated and therefore not deformed by Tet-system proteins.

[0029] Therefore, the present invention provides a liver cancer diagnostic kit comprising a primer pair for amplifying a fragment containing a CpG site of one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2.

[0030] In addition to the formulation, the composition and kit may also contain polymerase agarose, a buffer solution necessary for electrophoresis, and other similar materials.

[0031] Furthermore, the present invention provides a nucleic acid chip for liver cancer diagnosis on which a probe capable of hybridizing with a fragment containing a CpG site of one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 is immobilized.

[0032] In this invention, the term "nucleic acid" means oligonucleotides, nucleotides, polynucleotides, or fragments thereof, single-stranded or double-stranded DNA or RNA of genomic or synthetic origin, sense or antisense strands of DNA or RNA of genomic or synthetic origin, PNA (peptide nucleic acid), or substances of naturally occurring or synthetic origin in amounts of DNA or RNA. When the nucleic acid is RNA, it will be obvious to those ordinary skill in the art that deoxynucleotides A, G, C, and T are substituted with ribonucleotides A, G, C, and U, respectively.

[0033] Since methylation begins on the outer edge of a gene's regulatory site and progresses inward, detecting methylation on the outer edge of the regulatory site allows for early diagnosis of genes involved in cell transformation. Therefore, early diagnosis of cells potentially forming liver cancer is possible using the methylation gene markers. When genes that have been confirmed to be methylated in cancer cells are methylated in clinically or morphologically normal-looking cells, these normal-looking cells are actually undergoing cancerous transformation. Therefore, early diagnosis of liver cancer is possible by confirming the methylation of liver cancer-specific genes in normal-looking cells.

[0034] Furthermore, the present invention relates to the step of measuring the methylation level of the CpG site of one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 from a sample of a patient suspected of having liver cancer; and The present invention provides a method for diagnosing liver cancer, comprising the step of comparing the measured methylation level with the methylation level of the CpG site of the same gene in a normal control sample.

[0035] The methods for measuring the methylation level include PCR, methylation-specific PCR, and real-time methylation-specific PCR. The following methods may be selected, but are not limited to, time methylation-specific PCR, PCR using methylated DNA-specific binding proteins, methylation detection using methylation-sensitive restriction enzymes, quantitative PCR, DNA chips, pyrosequencing, and bisulfite sequencing.

[0036] Specifically, the methylation-specific PCR method involves treating the sample DNA with bisulfite, and then designing and using different types of primers depending on whether or not the CpG dinucleotide is methylated. If the primer binding site is methylated, the PCR proceeds using the methylated primer; if it is not methylated, the PCR proceeds using the normal primer. In other words, after treating the sample DNA with bisulfite, PCR is performed using two types of primers simultaneously, and then the results are compared.

[0037] Real-time methylation-specific PCR is a method that switches the methylation-specific PCR method to a real-time measurement method, where genomic DNA is treated with bisulfite and then methylated... This involves designing PCR primers that match the target DNA sequence and performing real-time PCR using these primers. In this case, there are two methods: one using a TanMan probe complementary to the amplified base sequence for detection, and another using Sybergreen for detection. Therefore, real-time methylation-specific PCR can selectively quantitatively analyze only methylated DNA. In this case, a standard curve is created using an in vitro methylated DNA sample, and for standardization, a gene without a 5'-CpG-3' sequence in its base sequence is amplified together with a negative control group, and the degree of methylation is quantitatively analyzed.

[0038] In a method for measuring the presence or absence of methylation using methylation-sensitive restriction enzymes, the methylation-sensitive restriction enzymes use CpG dinucleotides as their action site, and if this site is methylated, the enzyme cannot function. Therefore, when sample DNA is treated with a methylation-sensitive restriction enzyme and then amplified by PCR to include the enzyme target site, methylated sites are amplified by PCR without the restriction enzyme acting, while unmethylated normal sites are not amplified by PCR due to cleavage by the restriction enzyme. Thus, the presence or absence of methylation at a specific DNA site can be measured.

[0039] PCR or DNA chip methods using methylated DNA-specific binding proteins allow for the selective separation of methylated DNA because the protein, which specifically binds only to methylated DNA, is mixed with the DNA. Genomic DNA is mixed with the methylated DNA-specific binding protein, and then only the methylated DNA is selectively separated. These separated DNAs are amplified using PCR primers corresponding to the intron regions, and then the presence or absence of methylation is measured by agarose electrophoresis. Alternatively, the presence or absence of methylation can also be measured by quantitative PCR. Methylated DNA separated with the methylated DNA-specific binding protein can be labeled with a fluorescent dye and hybridized onto a DNA chip where complementary probes are accumulated to measure the presence or absence of methylation. Here, the methylated DNA-specific binding protein is not limited to MBD2bt.

[0040] Furthermore, pyrosequencing of DNA treated with bisulfite is based on the following principle: When methylation occurs at the CpG dinucleotide site, 5-methylcytosine (5-mC) is formed. This deformed base changes to uracil when treated with bisulfite. When DNA extracted from a sample is treated with bisulfite, if the CpG dinucleotide is methylated, it is preserved as cytosine, and the remaining unmethylated cytosine changes to uracil. Sequence analysis of bisulfite-treated DNA can preferably be performed using pyrosequencing. Detailed explanations of pyrosequencing are known in prior literature. [Ronaghi et al,Science 1998 Jul 17, 281(5375),363-365;Ronaghi et al, Analytical Biochemistry 1996 Nov 1,242(1),84-9;Ronaghi et al. Analytical Biochemistry 2000 Nov 15, 286(2):282-288;Nyr,P.Methods mol Biology 2007,373,114].

[0041] On the other hand, as a bisulfite-independent detection method using the Tet protein, it is also possible to detect the base at the methylation site by converting only methylated C to T using the Tet protein (see LIU, Yibin, et al., Nature Biotechnology volume 37, pages 424-429 (2019)). When methylation occurs at the CpG dinucleotide site, and cytosine is converted to 5-methylcytosine (5-mC), if the Tet (ten-eleven translocation) protein is methylated, then when processing the Tet (ten-eleven translocation) protein, if the CpG dinucleotide is methylated, The cytosine that is converted to racil and not methylated is conserved. Sequence analysis of Tet-treated DNA is not limited to pyrosequencing, but can also be performed using methods such as methylation-sensitive PCR (MSP), microarrays, and next-generation sequencing (NGS).

[0042] Preferably, the liver cancer diagnostic method of the present invention may be carried out by a method comprising: a) obtaining a sample from an individual; b) obtaining genomic DNA from the sample; c) treating the obtained genomic DNA with a compound that deforms unmethylated cytosine bases; d) obtaining a PCR product by amplifying the treated DNA by PCR using primers that can amplify any one or more CpG sites selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2; and e) measuring the degree of methylation of the PCR product.

[0043] The acquisition of genomic DNA in step b) above can be carried out using phenol / chloroform extraction, SDS extraction (Tai et al., Plant Mol. Biol. Reporter. 8:297-303, 1990), CTAB separation (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980), or commercially available DNA extraction kits, which are commonly used in the industry.

[0044] In the present invention, the term "sample" refers to a broad range of bodily fluids, including all biological bodily fluids obtained from individuals, body fluids, cell lines, tissue cultures, etc., depending on the type of analysis performed. Methods for obtaining bodily fluids and tissue biopsies from mammals are generally well known, and in the present invention, the sample may preferably be selected from the group consisting of human-derived substances, including tissues, cells, blood, plasma, serum, feces, and urine. Since abnormal methylation changes in cancerous tissue show considerable similarity to methylation changes in genomic DNA obtained from biological samples such as cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, using the markers of the present invention has the advantage of enabling simple diagnosis using blood, bodily fluids, etc., for predicting the development of liver cancer.

[0045] Furthermore, the present invention provides the use of a formulation for measuring the methylation of one or more gene CpG sites selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2, for the preparation of a hepatocyte cancer diagnostic formulation. [Effects of the Invention]

[0046] As described above, hypermethylation of the CpG site of one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 is specifically observed in liver cancer. Therefore, liver cancer can be accurately and rapidly diagnosed, and even earlier, using the composition, kit, chip, or method according to the present invention. [Brief explanation of the drawing]

[0047] [Figure 1] Figure 1 shows the results of examining the methylation information of the FAM110A gene in a total of 33 cancer types. [Figure 2] Figure 2 shows the results of examining the methylation information of the FAR1 gene in a total of 33 cancer types. [Figure 3] Figure 3 shows the results of examining the methylation information of the VIM gene in a total of 33 cancer types. [Figure 4] Figure 4 shows the results of examining the methylation information of the LDHB gene in a total of 33 cancer types. [Figure 5] Figure 5 shows the results of examining the methylation information of the LIPE gene in a total of 33 cancer types. [Figure 6] Figure 6 shows the results of examining the methylation information of the INAFM1 gene in a total of 33 cancer types. [Figure 7] Figure 7 shows the results of examining the methylation information of the ATL1 gene in a total of 33 cancer types. [Figure 8] Figure 8 shows the results of examining the methylation information of the CELF6 gene in a total of 33 cancer types. [Figure 9] Figure 9 shows the results of examining the methylation information of the MTHFD2 gene in a total of 33 cancer types. [Figure 10] Figure 10 shows the results of examining the methylation information of the PAK1 gene in a total of 33 cancer types. [Figure 11]Figure 11 shows the results of examining the methylation information of the NXPE3 gene in a total of 33 cancer types. [Figure 12] Figure 12 shows the results of examining the methylation information of the SLC25A36 gene in a total of 33 cancer types. [Figure 13] Figure 13 shows the results of examining the methylation information of the VANGL2 gene in a total of 33 cancer types. [Figure 14] Figure 14 shows the results of confirming the diagnostic accuracy of hepatocellular carcinoma in a total of 14 genes selected according to the present invention. [Figure 15] Figure 15 shows the results of confirming the differences in methylation between tumor cell lines and non-tumor cell lines. [Figure 16] Figures 16a and 16b show the methylation levels of the FAR1, PAK1, ATL1, and LIPE genes in liver cancer tissue and normal surrounding tissue, after qMSP analysis, presented as ΔCt+10 values. [Figure 17] Figure 17 shows a comparative example, which confirms the methylation information of the GRASP gene. [Examples]

[0048] The following are preferred embodiments to aid in understanding the present invention. However, these embodiments are provided to facilitate understanding of the present invention and do not limit its scope.

[0049] Example 1: Selection of hepatocellular carcinoma-specific methylated genes To select methylated genes specifically found in hepatocellular carcinoma, a large-scale comparative study of methylation in cancerous and normal tissues obtained from cancer surgery in colorectal cancer patients was conducted using data from three large-scale methylation microarray chips (see Table 2). In this study, tumor tissue refers to cancerous tissue from hepatocellular carcinoma, and non-tumor tissue refers to non-tumor tissue. "Tissues" refers to tissues other than cancerous tissue, including normal liver tissue.

[0050] [Table 2]

[0051] To select hepatocellular carcinoma-specific methylated genes, DNA was extracted from various tissues, and the degree of methylation at the gene sites was confirmed using an Infinium Human Methylation 450 Beadchip microarray. DNA extracted from each tissue is transformed by bisulfite treatment. This alters the cytosine base depending on whether or not the DNA site is methylated. The probes used in this microarray experiment were specifically designed for methylation and unmethylation to confirm the alteration of cytosine bases at methylated sites in genes. This microarray experiment measures the degree of methylation of genes using approximately 450,000 (450k) probes that indicate the methylation sites of each gene. The results for each probe derived from the experiment are shown as β values. β values ​​range from 0 to 1, with values ​​closer to 1 indicating a higher degree of methylation in that gene region. To identify differentially methylated regions (DMRs) between the tumor and non-tumor groups, we used the Limma (Linear Models for Microarray Data) empirical Bayes t-test to identify gene sites showing statistically significant differences in methylation between the groups. The Limma method is known to be the least affected by outliers among several methylation statistical analysis methods used to identify differences between groups. Therefore, it is less affected by abnormal measurements from some samples and is a suitable method for finding cancer-specific markers. In this experiment, a smaller adjusted p-value derived by the Limma method was considered to indicate a significant methylation difference between the two groups. In particular, to explore tumor-specific methylation sites, we selected gene regions with significant differences in β values ​​between tumor and non-tumor groups, specifically those showing higher methylation in tumor tissue compared to non-tumor tissue, as candidate cancer-specific biomarkers. As a result of limma analysis in each of the three datasets, gene regions that showed significantly lower p-values ​​when comparing tumor groups compared to non-tumor groups, and exhibited large differences of 0.2 or more in inter-group β values, were selected as tumor-specific hypermethylated regions. This allowed us to select 1,777 gene regions out of approximately 450,000 gene regions that commonly showed tumor-specific hypermethylation across all datasets as candidate biomarkers.

[0052] Example 2: Selection of hepatocellular carcinoma-specific hypermethylated genes In the 1,777 biomarker gene sites identified in Example 1, the degree of methylation at each corresponding site in tumors other than hepatocellular carcinoma was examined and compared to identify hepatocellular carcinoma-specific gene sites among the biomarkers. DNA methylation 450k Analysis of the experimental results of the array revealed methylation information for genetic sites corresponding to 33 types of cancer. Of these, hepatocellular carcinoma (which we will call liver cancer) and the other 32 types of cancer showed significantly higher β values ​​in liver cancer compared to other cancers. We were able to confirm that liver cancer-specific methylation occurred in 42 out of 1,777 genetic sites.

[0053] The degree of methylation of the aforementioned gene in tumor tissue (cancer tissue of hepatocellular carcinoma) and non-tumor tissue (tissue other than cancer tissue, including normal liver tissue) as determined by the microarray experiment is the same as in Figure 15. The degree of methylation is shown as a β value, which is the result of each probe derived from the test. The β value ranges from 0 to 1, and a value closer to 1 indicates a higher degree of methylation of the corresponding gene site.

[0054] On the other hand, in the case of genetic sites where differences in methylation are observed when comparing tumor tissue from liver cancer with non-tumor tissue, methylation can also occur in other cancers besides liver cancer. In other words, liver cancer-specific methylation has not been confirmed.

[0055] For example, GRASP (General Receptor for Phosphoinositides 1) In the case of the associated scaffold protein gene, it was one of the sites where the largest difference in methylation between tumor and non-tumor tissue was observed among the 1,777 genetic sites identified in Example 1. As shown in Figure 16, high methylation was observed in liver cancer, and it was also confirmed that high methylation occurred in various cancers, including prostate cancer, colorectal cancer, and gastric cancer.

[0056] The 33 types of cancer mentioned above are as follows: Acute Myeloid Leukemia, Adrenocortical cancer, Bile Duct cancer, Breast cancer, Cervical Cancer, Colon cancer, Endometrioid Cancer, Esophageal Cancer, Glioblastoma, Head and neck cancer Kidney chromophobe, Kidney clear cell carcinoma, Kidney papillary cell carcinoma, Large B-cell lymphoma, Liver cancer, Lower Grade Glioma, Lung adenocarcinoma adenocarcinoma), melanoma, mesothelioma, ocular melanoma melanomas), Ovarian cancer, Pancreatic cancer, Brown cell tumor & Pheochromocytoma ganglioma, Prostate cancer, Rectal cancer, Sarcoma, Stomach cancer, Testicular cancer, Thymoma, Thyroid cancer cancer), uterine sarcoma (uterine carcinosarcoma).

[0057] Of these gene regions, those that were not pseudogenes, but located in CpG island regions, within + / - 2000 base pairs (2kb) of the gene's transcription start site (TSS), and on autosomes, were selected as hepatocellular carcinoma-specific hypermethylated genes. As a result, a total of 13 genes were selected, as shown in Table 3 below (see Figures 1-13).

[0058] [Table 3]

[0059] In particular, FAR1 (Figure 2), LIPE (Figure 5), MTHFD2 (Figure 10), and NXPE3 (Figure 12) showed clear specific hypermethylation compared to other cancerous liver cancers.

[0060] Example 3: Confirmation of liver cancer specificity of selected genes in cell lines To determine whether the 13 selected genes exhibit liver cancer-specific methylation that distinguishes them from other cancers, we utilized public databases and analyzed methylation patterns in 1,022 cancer cell lines derived from 14 major tissues. The data were obtained by performing Infinium Human Methylation 450 Beadchip microarray experiments according to the manufacturer's standardized methylation analysis process for DNA extracted from each cell line.

[0061] The results of the experiment were obtained by measuring the degree of gene methylation through approximately 450,000 probes, as in Example 1 above, and the methylation value of each probe is shown as a β value. The β value ranges from 0 to 1, and the closer it is to 1, the higher the degree of methylation of the corresponding gene site is considered to be. The 14 tissues mentioned above are as follows: respiratory gastrointestinal tract (aerodigestive) Tracts, blood, bones, breasts, digestive system, kidneys, lungs, nervous system, pancreas, skin, soft tissues, thyroid gland, urogenital system, and other tissues.

[0062] To confirm liver cancer-specific methylation of the 14 selected genes, methylation data from 1,022 cell lines were broadly classified into liver cancer cell lines (n=19) and non-liver cancer cell lines (n=1,003).

[0063] To identify differential methylation sites (DMRs) between the two classified groups, we used the Limma empirical Bayesian t-test to identify gene regions showing statistically significant differences in methylation between the groups.

[0064] [Table 4]

[0065] In particular, in the case of FAR1, LIPE, MTHFD2, and NXPE3, which previously showed clear specific hypermethylation in actual patient samples compared to other cancers in hepatocellular carcinoma, analysis using cell lines also confirmed their hepatocellular carcinoma specificity, as they consistently exhibited lower corrected p-values ​​in hepatocellular carcinoma cell lines compared to other cancer cell lines. In addition, genes such as LDHB and PAK1 were confirmed to show clear methylation in hepatocellular carcinoma cell lines compared to other cell lines.

[0066] Example 4: Evaluation of diagnostic performance of candidate liver cancer diagnostic markers To confirm the usefulness of the selected genes as diagnostic markers in hepatocellular carcinoma, the accuracy of hepatocellular carcinoma diagnosis based on methylation levels was evaluated.

[0067] To evaluate the accuracy of a diagnosis, sensitivity and specificity are used. By calculating the sensitivity and specificity values ​​for possible cut-off values ​​of consecutive diagnostic test measurements, a Receiver Operating Characteristic (ROC) curve can be shown, illustrating the change in sensitivity and specificity with respect to the cut-off value. Diagnostic accuracy is measured by the area under the ROC curve. The ROC curve (AUC) can measure this. The AUC value is between 0.5 and 1, and a higher value indicates higher diagnostic accuracy. A value of 1 indicates that the test result is completely accurate, while a value of 0.5 indicates that it is equivalent to a random result.

[0068] Analysis using a methylation dataset, which collected data on cancer classification accuracy based on methylation levels between non-tumor and tumor tissues using selected genes, revealed that all selected genes had AUC values ​​of 0.860 or higher, demonstrating high diagnostic accuracy and confirming their usefulness in the diagnosis of hepatocellular carcinoma. (Figure 14)

[0069] Example 5: Measurement of methylation of selected genes on the qMSP basis of liver cancer tissue. Of the 13 selected genes, four genes—FAR1, PAK1, ATL1, and LIPE—which showed a large difference in methylation levels between non-tumor and tumor tissues and had high average methylation levels in tumor tissue, underwent additional validation studies using cancer tissue. To confirm liver cancer-specific methylation of the four selected genes in cancer tissue, the difference in methylation between cancer and non-tumor tissues was measured using methylation-specific PCR (quantitative methylation-specific PCR, qMSP) technique. For this purpose, genomic DNA was isolated from cancer tissue and surrounding tissue pairs of 15 liver cancer patients (five patients from each stage of cancer, from stage 2 to stage 4). After bisulfite treatment, amplification of specific gene sites and methylation levels for FAR1, PAK1, ATL1, and LIPE were observed according to a generalized qMSP experimental method.

[0070] Furthermore, the ACTB gene, which is unrelated to methylation, was used as an internal standard to specifically bind to and amplify the genetic region converted to bisulfite, and to standardize the amplified value of that region. The methylation level obtained by amplified DNA converted to the aforementioned bisulfite by PCR is expressed as ΔCt+10, which is a value corrected by the ACTB Ct (Cycle of threshold) value used as an internal standard. ΔCt+10 is defined as follows: ΔCt+10 = (Ct value of ACTB gene - Ct value of target gene) + 10

[0071] As shown in Figures 16a and 16B, methylation of the FAR1, ATL1, PAK1, and LIPE genes showed relatively high ΔCt+10 values ​​in colorectal cancer tissue, regardless of disease stage, compared to normal tissue surrounding the cancer. This confirmed that the four genes FAR1, ATL1, PAK1, and LIPE were hypermethylated in liver cancer. This result demonstrates that the methylation of the selected FAR1, ATL1, PAK1, and LIPE genes is effective as a biomarker for the diagnosis of liver cancer, especially for early diagnosis. These results indicate that the selected genes can also be used in the diagnosis of liver cancer. [Industrial applicability]

[0072] As described above, hypermethylation of the CpG site of one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 is specifically observed in liver cancer. Therefore, the compositions, kits, chips, or methods according to the present invention can be used not only to accurately and rapidly diagnose liver cancer, but also to enable early diagnosis. [Prior art documents] [Patent Documents]

[0073] [Patent Document 1] International Publication No. 01 / 26536 [Patent Document 2] U.S. Patent Application Publication 2003 / 0148326, Specification A1 [Non-patent literature]

[0074] [Non-Patent Document 1] Singleton et al.DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOTY(2th ed. 1994) [Non-Patent Document 2] THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY(Walkered., 1988) [Non-licensed document 3] Hale&Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY

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Non-licensed literature 9

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Claims

1. A diagnostic composition for liver cancer comprising a preparation for measuring the CpG site methylation level of the PAK1 gene, The composition is configured to diagnose liver cancer by detecting methylation of the CpG site of the PAK1 gene, which is methylated in liver cancer, using a human-derived biological sample, wherein the CpG site is the region 77122736–77123088 on chromosome 11, expressed according to The February 2009 Human reference sequence (GRCh37). A composition for diagnosing liver cancer.

2. The composition according to claim 1, further comprising a formulation for measuring the methylation level of a CpG site of any one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, NXPE3, SLC25A36, and VANGL2.

3. The composition according to claim 1 or claim 2, characterized in that the CpG site is located within + / - 2000 base pairs (2 kb) from the transcription start site of the gene.

4. A formulation for measuring the methylation level of the CpG site of the aforementioned gene is Compounds that deform unmethylated cytosin or methylated cytosin bases; and (a) A primer specific to the methylated sequence of the CpG region of the gene; (b) A primer specific to the unmethylated sequence of the CpG region of the gene, (c) Both (a) and (b) The composition according to any one of claims 1 to 3, characterized by comprising at least one primer selected from the group consisting of the following.

5. The compound that deforms the unmethylated cytosine base is bisulfite or a salt thereof, and the compound that deforms the methylated cytosine base is Tet The composition according to claim 4, characterized in that it is a protein.

6. (a) A primer pair for amplifying a fragment containing a CpG site of the PAK1 gene, wherein the CpG site is located within + / - 2000 base pairs (2 kb) from the transcription start site of the gene. (b) Formulations for measuring the methylation level of amplified fragments, and (c) Buffer solution suitable for amplification and measurement reactions A liver cancer diagnostic kit that includes, The kit is configured to diagnose liver cancer by detecting methylation of the CpG region of the PAK1 gene, which is methylated in liver cancer, using a human-derived biological sample, wherein the CpG region is the region 77122736–77123088 on chromosome 11, expressed according to The February 2009 Human reference sequence (GRCh37). A kit for diagnosing liver cancer.

7. The kit according to claim 6, further comprising a primer pair for amplifying a fragment containing a CpG site of any one or more genes selected from the group consisting of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, NXPE3, SLC25A36, and VANGL2.

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