Compositions for diagnosing liver cancer by using cpg methylation changes in specific genes and uses thereof

Abstract

This invention relates to a composition, kit, nucleic acid chip, and method for diagnosing liver cancer by detecting the methylation level of CpG sites in one or more genes selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2. This invention enables accurate and rapid diagnosis of liver cancer, and also allows for early diagnosis.

Description

[0001] This application is a divisional application of the invention patent application filed on October 8, 2020, with application number 202080086538.2 and invention title "Composition for diagnosing liver cancer by using changes in CPG methylation in a specific gene and its use therein". Technical Field

[0002] This invention relates to a composition, kit, nucleic acid chip, and method for diagnosing liver cancer by detecting the methylation level of CpG sites selected from the following genes: FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2. Background Technology

[0003] This application claims priority to Korean Patent Application No. 2019-0127218, filed on October 14, 2019, the entire contents of which are incorporated herein by reference.

[0004] Liver cancer is one of the most common cancers worldwide. In South Korea, the mortality rate from liver cancer is extremely high, with 23 deaths per 100,000 people. Approximately 10% of all deaths in South Korea are related to hepatitis, cirrhosis, and liver cancer. Liver cancer is difficult to diagnose early because it often presents with no noticeable symptoms. Generally, because liver cancer is usually discovered at an advanced stage where it cannot be properly treated, treatment options are very limited and the prognosis is poor. Since the prognosis of liver cancer varies greatly depending on the cancer's progression at diagnosis, early detection is crucial for improving the survival rate of liver cancer patients.

[0005] On the other hand, epigenetics is the field that studies gene expression regulation without altering the DNA base sequence. Epigenetics studies the regulation of gene expression through epigenetic variations, such as DNA methylation, acetylation, phosphorylation, and ubiquitination of miRNAs or histones.

[0006] DNA methylation is one of the most studied epigenetic variations. Epigenetic variations can lead to gene function abnormalities and changes in tumor cells. Therefore, DNA methylation is associated with the expression (or suppression and induction) of disease-regulating genes in cells, and methods for diagnosing cancer by measuring DNA methylation have recently been proposed. In particular, since cancer-specific methylation occurs even earlier in precancerous tissues, the detection of cancer-specific methylation is highly likely to be used for cancer diagnosis.

[0007] Therefore, it is necessary to develop effective liver cancer-specific methylation biomarkers that can predict the risk of liver cancer.

[0008] Invention disclosure Technical issues Therefore, the inventors discovered that the CpG sites of specific genes in liver cancer are in a hypermethylated state, and developed compositions, kits, nucleic acid chips and methods that can diagnose liver cancer by detecting methylation levels, and then completed the present invention.

[0009] One object of the present invention is to provide a composition for diagnosing liver cancer, comprising a preparation for measuring the methylation level of CpG sites of a specific gene.

[0010] Another object of the present invention is to provide a kit for diagnosing liver cancer, comprising a PRC primer pair for amplifying a fragment including a CpG site of a specific gene and sequencing primers for pyrosequencing the PCR product amplified by said primer pair.

[0011] Another object of the present invention is to provide a nucleic acid chip for diagnosing liver cancer, wherein probes are immobilized that are capable of hybridizing with fragments including CpG sites of specific genes under stringent conditions.

[0012] Another object of the present invention is to provide a method for diagnosing liver cancer, comprising measuring and comparing the methylation levels of CpG sites of specific genes from different samples.

[0013] Another object of the present invention is to provide the use of preparations for measuring the methylation level of CpG sites in genes in the preparation of preparations for diagnosing liver cancer.

[0014] Technical solution To achieve the stated objective, a composition for diagnosing liver cancer is provided, comprising preparations for measuring the methylation level of CpG sites selected from one or more of the following genes: FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2.

[0015] For another purpose, a kit for diagnosing liver cancer is provided, comprising primer pairs for amplifying fragments including CpG sites selected from any one or more of the following genes: FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2.

[0016] To achieve another objective, a nucleic acid chip for diagnosing liver cancer is provided, the nucleic acid chip being immobilized with probes capable of hybridizing with fragments including CpG sites selected from any one or more of the following genes: FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2.

[0017] To achieve another objective, a method for diagnosing liver cancer is provided, comprising measuring the methylation levels of CpG sites selected from one or more of the following genes in a sample from a patient suspected of having liver cancer: FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2; and The measured methylation levels were compared with the methylation levels of the CpG sites of the same gene in normal control samples.

[0018] For yet another purpose, the use of preparations for measuring the methylation level of CpG sites selected from one or more of the following genes in the preparation of preparations for diagnosing liver cancer is provided.

[0019] 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 general definitions of the various terms used in this specification for those skilled in the art: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOTY (2nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walkered., 1988); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY.

[0020] The present invention will be described in detail below.

[0021] This invention relates to a composition for diagnosing liver cancer, comprising preparations for measuring the methylation level of CpG sites selected from one or more of the following genes: FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2.

[0022] As used herein, the term "methylation" refers to the attachment of a methyl group to a base that constitutes DNA. Preferably, in this invention, methylation refers to whether cytosine at a specific CpG site of a particular gene is methylated. When methylation occurs, the binding of transcription factors is disrupted, thereby suppressing the expression of the particular gene. Conversely, when unmethylation or hypomethylation occurs, the expression of the particular gene increases.

[0023] In mammalian cell genomic DNA, in addition to A, C, G, and T, there is a fifth base called 5-methylcytosine (5-mC), with its methyl group attached to the fifth carbon of the cytosine ring. Methylation of 5-methylcytosine occurs only at the C of a CG dinucleotide called CpG (5'-mCG-3'), and methylation of CpG represses the expression of alu or transposons and genomic repetitive sequences. Furthermore, because the 5-mC of CpG is readily deaminated and converted to thymine (T), CpG is the most frequently occurring site of epigenetic changes in mammalian cells.

[0024] As used herein, the term "measurement of methylation level" refers to the measurement of the methylation level at CpG sites of any or more genes selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2, and the measurement of methylation level according to bisulfite-treated or bisulfite-free detection methods. Methylation level can be measured by methylation-specific PCR, such as methylation-specific polymerase chain reaction (MSP), real-time methylation-specific polymerase chain reaction (PCR), PCR using methylation DNA-specific binding proteins, or quantitative PCR. Optionally, methylation level can be measured by automated sequencing such as pyrosequencing and bisulfite sequencing, but is not limited thereto. Furthermore, methylation level can be measured using a detection method employing the 10-11 translocation protein (TET protein) as a bisulfite-free detection method (see Nature Biotechnology, volume 37, pages 424-429 (2019)). TET protein is an enzyme that acts on DNA and participates in base chemical changes. When treated with bisulfite, all C bases except methylated C are converted to T bases. However, in Tet protein, only methylated C is converted to T for effective detection.

[0025] Preferably, the CpG site selected from any one or more genes of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 refers to a CpG site on the DNA of the gene. The DNA of a gene is a concept encompassing all the structural units required for gene expression and operatively interconnected, and may include, for example, promoter regions, open reading frames (ORFs), and terminator regions. Therefore, the CpG site of any one or more genes of FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 is located in the promoter region, open reading frame (ORF), or terminator region of the corresponding gene.

[0026] Preferably, in this invention, measuring the methylation level of CpG sites in any one or more genes selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 can mean measuring the methylation level of cytosine in the CpG sites of the genes shown in Table 1 below.

[0027] [Table 1]

[0028] In this invention, the CpG site of any one or more genes selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36 and VANGL2 is located within + / - 2000 bases (2 kb) from the transcription start site (TSS).

[0029] In this invention, the base sequences of human genome chromosomal regions are expressed based on the February 2009 human reference sequence (GRCh37). However, the expression of specific sequences in human genome chromosomal regions can change slightly with updated genome sequence studies, and the expression of human genome chromosomal regions in this invention can vary accordingly. Therefore, in the human genome chromosomal regions expressed according to the February 2009 human reference sequence (GRCh37) of this invention, since the human reference sequence has been updated since the application date of this invention, even if the expression of the human genome chromosomal regions is changed and differs from the present, the scope of this invention obviously affects the changed human genome chromosomal regions. Those skilled in the art will readily see these changes.

[0030] In this invention, the preparations used to measure the methylation level of CpG sites may include compounds that modify cytosine bases or methylation-sensitive restriction enzymes, primers specific to methylated allele sequences selected from any one or more of the following genes: FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2, and primers specific to unmethylated allele sequences.

[0031] The compound modifying the cytosine base is a compound that modifies unmethylated or methylated cytosine, and may be a bisulfite or a salt thereof, preferably sodium bisulfite modifying unmethylated cytosine, or a TET protein modifying methylated cytosine, but is not limited thereto. Methods for detecting whether CpG sites are methylated by modifying cytosine bases are well known in the art (WO 01 / 26536; US 2003 / 0148326A1).

[0032] Furthermore, methylation-sensitive restriction enzymes can be restriction enzymes that specifically detect methylation at CpG sites, and can be restriction enzymes containing CG as the recognition site. Examples include SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, etc., but are not limited to these. Depending on whether the C site of the restriction enzyme recognition site is methylated or unmethylated, the cleavage of the restriction enzyme will vary, and can be detected by PCR or Southern blotting analysis. Methylation-sensitive restriction enzymes other than those described above are also well known in the art.

[0033] The primers may include primers specific to the methylated allele sequences of any one or more genes selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2, as well as primers specific to the unmethylated allele sequences.

[0034] In this invention, the term "primer" refers to a short nucleic acid sequence having a short, free 3-terminal hydroxyl group, and a short nucleic acid sequence capable of forming a base pair with a complementary template and serving as the starting point for copying the template strand. Under appropriate buffer and temperature conditions, in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleosides of triphosphate, the primers can initiate DNA synthesis. Furthermore, the primers are sense and antisense nucleic acids having sequences of 7 to 50 nucleotides, and additional features can be incorporated without altering the fundamental properties of the primers used as DNA synthesis initiation sites.

[0035] The primers of the present invention may preferably be designed based on the sequence of a specific CpG site for analyzing methylation, and more preferably may be at least one primer pair selected from the following: primer pair capable of specifically amplifying methylated cytosine not modified by bisulfite, primer pair capable of specifically amplifying unmethylated cytosine modified by bisulfite, primer pair capable of specifically amplifying methylated cytosine modified by Tet-based proteins, and primer pair capable of specifically amplifying unmethylated cytosine not modified by Tet-based proteins.

[0036] Therefore, the present invention provides a kit for diagnosing liver cancer, comprising primer pairs for amplifying fragments of CpG sites from any one or more genes selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2.

[0037] In addition to the prepared material, the composition and kit may also include polymerase, agarose, buffer, etc., required for electrophoresis.

[0038] In addition, the present invention provides a nucleic acid chip for diagnosing liver cancer, which is immobilized with probes capable of hybridizing with CpG sites of any or more genes selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36 and VANGL2.

[0039] In this invention, the term "nucleic acid" refers to oligonucleotides, nucleotides, polynucleotides or fragments thereof, single-stranded or double-stranded genomic or synthetically derived DNA or RNA, sense or antisense strands of genomic or synthetically derived DNA or RNA, and peptide nucleic acids (PNAs) or naturally or synthetically derived DNA or RNA analogs. If the nucleic acid is RNA, those skilled in the art will understand that ribonucleotides A, G, C, and T are substituted for deoxynucleotides A, G, C, and U, respectively.

[0040] Since methylation begins outside the regulatory site of a gene and proceeds into its interior, genes involved in cell transformation can be diagnosed at an early stage by detecting methylation outside the regulatory site.

[0041] Therefore, methylation gene markers can be used for early diagnosis of cells that may develop into liver cancer. When genes methylated in cancer cells are confirmed to be methylated in clinically or morphologically normal cells, those seemingly normal cells are becoming cancerous. Thus, liver cancer can be diagnosed early by confirming the methylation of liver cancer-specific genes in seemingly normal cells.

[0042] Furthermore, this invention provides a method for diagnosing liver cancer, comprising measuring the methylation level of CpG sites in one or more genes selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 from samples from suspected liver cancer patients; and The measured methylation levels were compared with the methylation levels of the CpG sites of the same gene in normal control samples.

[0043] Methods for measuring methylation levels may be selected from, but are not limited to, PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR using methylation DNA-specific binding proteins, measuring the presence of methylation using methylation-sensitive restriction enzymes, quantitative PCR, DNA microarrays, pyrosequencing, and bisulfite sequencing.

[0044] Specifically, methylation-specific PCR involves treating sample DNA with bisulfite and then designing and using different types of primers based on whether the CpG dinucleotides are methylated. If the primer binding site is methylated, methylated primers are used for PCR; otherwise, normal primers are used. In other words, this method involves treating sample DNA with bisulfite, performing PCR with two types of primers simultaneously, and comparing the results.

[0045] Real-time methylation-specific PCR converts the methylation-specific PCR method into a real-time measurement method. This involves treating genomic DNA with bisulfite, designing PCR primers corresponding to methylation, and using these primers for real-time PCR. Two methods are available: detection using TanMan probes complementary to the amplified base sequence, and detection using Sybergreen. Therefore, real-time methylation-specific PCR can selectively quantify only methylated DNA. In this case, the method uses an in vitro methylated DNA sample to prepare a standard curve and uses genes lacking the 5'-CpG-3' sequence in their amplified base sequences as a negative control for standardization to quantify methylation levels.

[0046] In methods for measuring methylation using methylation-sensitive restriction enzymes, the methylation-sensitive restriction enzyme uses a CpG dinucleotide as its action site, and this site does not function as an enzyme when methylated. Therefore, when sample DNA is treated with a methylation-sensitive restriction enzyme and then amplified by PCR to include the enzyme target site, the restriction enzyme is inactive in the presence of methylation, but amplification occurs via PCR. However, unmethylated normal sites are cleaved by the restriction enzyme and not amplified by PCR, thereby measuring whether a specific DNA site is methylated.

[0047] In PCR or DNA microarray methods using methylation DNA-specific binding proteins, when a protein that specifically binds to methylation DNA is mixed with DNA, the protein binds only specifically to methylation DNA, thus selectively isolating only methylation DNA. After mixing genomic DNA with a methylation DNA-specific binding protein, only methylation DNA is selectively isolated. This is done by amplifying these isolated DNAs using PCR primers corresponding to intron sites, and then measuring whether methylation has occurred by agarose gel electrophoresis. Furthermore, methylation can be measured by quantitative PCR, and by labeling the methylation DNA isolated by the methylation DNA-specific binding protein with a fluorescent dye and hybridizing it to a DNA microarray integrated with complementary probes to measure methylation. Here, the methylation DNA-specific binding protein is not limited to MBD2bt.

[0048] Furthermore, pyrosequencing of bisulfite-treated DNA is based on the following principle: When CpG dinucleotide sites are methylated, 5-methylcytosine (5-mC) is formed, and the modified base is converted to uracil after bisulfite treatment. If the CpG dinucleotides were already methylated when the DNA extracted from the sample was treated with bisulfite, the CpG dinucleotides are retained as cytosine, and the remaining unmethylated cytosine is converted to uracil. Sequencing of bisulfite-treated DNA can preferably be performed using pyrosequencing methods. Detailed descriptions of pyrosequencing are known in the prior art [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].

[0049] On the other hand, by using a bisulfite-free detection method for Tet protein, only methylated C of Tet protein can be converted to T to detect the bases at methylation sites (see LIU, Yibin, et al., Nature Biotechnology volume 37, pages 424-429 (2019)).

[0050] When methylation occurs at the CpG dinucleotide site, resulting in the formation of cytosine as 5-methylcytosine (5-mC), the CpG dinucleotide is methylated to uracil upon treatment with the 10-11 translocation (Tet) protein, while the unmethylated cytosine is retained. Sequencing of Tet-treated DNA is not limited to pyrosequencing methods; it can also be performed using methods such as methylation-sensitive PCR (MSP), microarrays, and next-generation sequencing (NGS).

[0051] Preferably, the method for diagnosing liver cancer according to the present invention can be performed by a method characterized by comprising the following steps: a) obtaining a sample from a subject, b) obtaining genomic DNA from the sample, c) treating the obtained genomic DNA with a compound that modifies unmethylated cytosine bases, d) obtaining a PCR product by PCR amplification of the treated DNA using primers capable of amplifying any or more CpG sites selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2, and e) measuring the methylation level of the PCR product.

[0052] In step b), genomic DNA can be obtained using methods commonly used in the art, such as phenol / chloroform extraction, SDS extraction (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), CTAB separation (CetylTrimethyl Ammonium Bromide; Murray et al., Nuc) Res., 4321-4325, 1980), or commercially available DNA extraction kits.

[0053] In this invention, the term "sample" refers to a broad range of bodily fluids, including all biological fluids, body fluids, cell lines, tissue cultures, etc., obtained from an individual, depending on the type of analysis to be performed. Methods for obtaining bodily fluids and tissue biopsies from mammals are generally well-known, and in this invention, the sample may preferably be selected from human derivatives, including tissues, cells, blood, plasma, serum, feces, and urine. Because abnormal methylation changes in cancerous tissue show significant similarities to methylation changes in the genomic DNA of biological samples such as cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, the use of biomarkers according to this invention offers the advantage of easy diagnosis via blood, bodily fluids, etc., for predicting the occurrence of liver cancer.

[0054] Furthermore, the present invention provides the use of preparations for measuring the methylation level of CpG sites of any one or more genes selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 in the preparation of preparations for the diagnosis of liver cancer.

[0055] Beneficial effects As described above, since hypermethylation of CpG sites in any or more genes selected from FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2 occurs particularly in liver cancer, the compositions, kits, chips, or methods of the present invention can accurately and rapidly diagnose liver cancer, as well as its early diagnosis.

[0056] Attached Figure Description Figure 1 The results confirmed the methylation information of the FAM110A gene in a total of 33 cancer types.

[0057] Figure 2 The results confirmed the methylation information of the FAR1 gene in a total of 33 cancer types.

[0058] Figure 3 The results confirmed the methylation information of the VIM gene in a total of 33 cancer types.

[0059] Figure 4 The results confirmed the methylation information of the LDHB gene in a total of 33 cancer types.

[0060] Figure 5 The results confirmed the methylation information of the LIPE gene in a total of 33 cancer types.

[0061] Figure 6 The results confirmed the methylation information of the INAFM1 gene in a total of 33 cancer types.

[0062] Figure 7 The results confirmed the methylation information of the ATL1 gene in a total of 33 cancer types.

[0063] Figure 8 The results confirmed the methylation information of the CELF6 gene in a total of 33 cancer types.

[0064] Figure 9 The results confirmed the methylation information of the MTHFD2 gene in a total of 33 cancer types.

[0065] Figure 10 The results confirmed the methylation information of the PAK1 gene in a total of 33 cancer types.

[0066] Figure 11 The results confirmed the methylation information of the NXPE3 gene in a total of 33 cancer types.

[0067] Figure 12The results confirmed the methylation information of the SLC25A36 gene in a total of 33 cancer types.

[0068] Figure 13 The results confirmed the methylation information of the VANGL2 gene in a total of 33 cancer types.

[0069] Figure 14 This confirms the accuracy of hepatocellular carcinoma diagnosis based on a total of 14 genes selected according to the present invention.

[0070] Figure 15 This is the result of confirming the differences in methylation between tumor tissue (tumor) cell lines and non-tumor tissue (other) cell lines.

[0071] Figures 16A and 16B show the methylation levels of FAR1, PAK1, ATL1, and LIPE genes after qMSP in hepatocellular carcinoma tissue and normal adjacent tissue, expressed as ΔCt + 10 values.

[0072] Figure 17 This is the result confirming the methylation information of the GRASP gene used as a comparative example.

[0073] Invention Model Preferred embodiments will be presented below to aid in understanding the invention. However, the following embodiments are provided merely for a better understanding of the invention, and the scope of the invention is not limited to these embodiments.

[0074] Example 1: Screening for Hepatocellular Carcinoma-Specific Methylation Genes To screen for methylation genes specifically found in hepatocellular carcinoma, a large-scale comparative study of methylation between cancerous and normal tissues obtained from colorectal cancer patients was conducted using data from three large-scale methylation microarrays (see Table 2). In this study, tumor tissue refers to cancerous tissue from hepatocellular carcinoma, and non-tumor tissue refers to tissues other than cancerous tissue, including normal liver tissue.

[0075] [Table 2]

[0076] To screen for hepatocellular carcinoma-specific methylation genes, DNA was extracted from each tissue, and the methylation level of the gene regions was confirmed using the Infinium Human Methylation 450 microbead array.

[0077] DNA extracted from each tissue was transformed by bisulfite treatment. In this way, cytosine bases were modified according to the methylation of DNA regions. Probes used in the microarray experiments were specifically designed for methylation and unmethylation to confirm whether cytosine bases in methylated regions of the gene were modified.

[0078] The microarray experiments measured gene methylation levels using approximately 450,000 (450 k) probes representing methylated regions of each gene, with the results from each probe presented as a β value. The β value ranges from 0 to 1, and a value closer to 1 indicates a higher methylation level in the corresponding gene region.

[0079] To identify differentially methylated regions (DMRs) between the tumor and non-tumor groups, gene regions with statistically significant differences in methylation between groups were identified using the empirical Bayes t-test and the microarray data linear model (Limma) method.

[0080] The Limma method is known to be least affected by outliers among several statistical methylation analyses used to identify differences between groups. Therefore, it is a suitable method for discovering cancer-specific biomarkers because it is less affected by outliers in certain samples. In our experiments, a significant difference in methylation between the two groups was determined by a decrease in the adjusted p-value obtained using the Limma method.

[0081] In particular, in order to find tumor-specific methylation regions, gene regions with significantly different β values ​​between the tumor group and the non-tumor group were selected as cancer-specific candidate biomarkers.

[0082] As a result, limma analysis performed on all three datasets showed that, compared to the non-tumor group, gene regions with significantly lower p-values ​​and larger β-values ​​(0.2 or greater) were selected as tumor-specific hypermethylated regions when comparing the tumor group. Consequently, out of approximately 450,000 gene regions, 1,777 gene regions exhibiting tumor-specific hypermethylation that were common across all datasets were selected as candidate biomarkers.

[0083] Example 2: Screening for Hepatocellular Carcinoma-Specific Hypermethylation Genes In Example 1, among the 1,777 gene regions identified as biomarkers, the methylation levels of each corresponding region in tumors other than hepatocellular carcinoma were identified and compared to find liver cancer-specific gene regions among the biomarkers. By analyzing the DNA methylation 450k array test results from the public cancer gene database The Cancer Genome Atlas (TCGA), methylation information of gene regions corresponding to 33 cancer types was confirmed. Specifically, by comparing hepatocellular carcinoma (referred to as liver cancer) with 32 other cancers, gene regions with significantly high β values ​​in liver cancer were identified, confirming liver cancer-specific methylation in 42 of the 1,777 gene regions.

[0084] Microarray experiments targeting genes were performed on tumor tissue (hepatocellular carcinoma tissue) and non-tumor tissue (including normal liver tissue and other tissues excluding cancerous tissue). The methylation levels of the genes were measured as follows: Figure 15 As shown. At the methylation level, the results of each probe obtained through experiments are represented as a β value, with β values ​​ranging from 0 to 1. It has been determined that the closer the β value is to 1, the higher the methylation level of the corresponding gene region.

[0085] Furthermore, given the observed differences in methylation in gene regions when comparing tumor and non-tumor tissues of liver cancer, methylation may occur in cancers other than liver cancer. In other words, liver cancer-specific methylation has not been confirmed.

[0086] For example, the general receptor for the phosphoinositol 1-associated scaffold protein (GRASP) gene is one of the regions among the 1,777 gene regions identified in Example 1 where the methylation differences between tumor and non-tumor tissues were most pronounced. However, as shown in Figure 16, hypermethylation was confirmed in liver cancer, and also in various cancer types including prostate cancer, rectal cancer, and gastric cancer.

[0087] The 33 types of cancer are as follows: acute myeloid leukemia, adrenocortical carcinoma, bile duct carcinoma, breast cancer, cervical cancer, colon cancer, endometrioid carcinoma, esophageal cancer, glioblastoma, head and neck cancer, chromophobe renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, large B-cell lymphoma, liver cancer, low-grade glioma, lung adenocarcinoma, melanoma, mesothelioma, ocular melanoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, sarcoma, stomach cancer, testicular cancer, thymoma, thyroid cancer and uterine carcinosarcoma.

[0088] Among these gene regions, those not pseudogenes, located at CpG island sites, within 2000 base pairs (2 kb) of the transcription start site (TSS), and situated on autosomes, were selected as hepatocellular carcinoma-specific hypermethylated genes. The results are shown in Table 3 below; a total of 13 genes were selected (see Table 3). Figures 1 to 13 ).

[0089] [Table 3]

[0090] In particular, compared with other cancer types, FAR1 ( Figure 2 ), LIPE Figure 5 ), MTHFD2 ( Figure 10 ) and NXPE3 ( Figure 12 It showed significant specific hypermethylation for liver cancer.

[0091] Example 3: Confirmation of the liver cancer specificity of the selected gene in the cell line To confirm whether the 13 selected genes exhibited liver cancer-specific methylation distinct from other cancers, methylation patterns from 1,022 cancer cell lines from 14 tissues were analyzed using a public database. For the corresponding data, DNA extracted from each cell line was subjected to Infinium Human Methylation 450 microbead array experiments according to the manufacturer's standardized methylation analysis test procedures.

[0092] In the experimental results, as in Example 1, the methylation level of the gene was measured using approximately 450,000 probes, and the methylation value for each probe was expressed as a β value. β values ​​range from 0 to 1, and it was determined that the closer the β value is to 1, the higher the methylation level of the corresponding gene region.

[0093] The 14 tissues are as follows: upper respiratory and digestive tract, blood, bones, mammary glands, digestive system, kidneys, lungs, nervous system, pancreas, skin, soft tissues, thyroid gland, urogenital system, and other tissues.

[0094] To confirm liver cancer-specific methylation of 14 selected genes, methylation data from 1,022 cell lines were primarily divided into liver cancer cell lines (n = 19) and non-liver cancer cell lines (n = 1,003).

[0095] To identify differentially methylated regions (DMRs) between two taxa, gene regions with statistically significant methylation differences between the groups were identified using empirical Bayesian t-tests and the Limma method for microarray data.

[0096] [Table 4] Methylation differences of selected genes between hepatocellular carcinoma cell lines and non-hepatocellular carcinoma cell lines

[0097] In particular, FAR1, LIPE, MTHFD2, and NXPE3 showed significant specific hypermethylation in liver cancer compared to other cancer types in actual patient samples, even when analyzed using cell lines, confirming that the liver cancer cell lines had very low adjusted p-values ​​specific to liver cancer compared to other cancer cell lines. Furthermore, genes such as LDHB and PAK1 showed significant methylation in the liver cancer cell lines compared to other cell lines.

[0098] Example 4: Diagnostic performance evaluation of candidate markers for liver cancer diagnosis To confirm the usefulness of the selected genes as diagnostic markers for hepatocellular carcinoma, the accuracy of diagnosing hepatocellular carcinoma based on methylation levels was evaluated.

[0099] To assess diagnostic accuracy, sensitivity and specificity are used. Receiver operating characteristic (ROC) curves can be plotted as changes in sensitivity and specificity based on the cutoff values ​​by calculating the sensitivity and specificity values ​​for consecutive diagnostic test measurements at feasible cutoff values. Diagnostic accuracy can be measured by the area under the ROC curve (AUC). An AUC value between 0.5 and 1 is considered to indicate higher diagnostic accuracy. An AUC value of 1 indicates a perfectly accurate test result, while an AUC value of 0.5 indicates that the diagnostic result is identical to a randomized result.

[0100] As a result of analyzing the accuracy of cancer classification based on methylation levels between non-tumor and tumor tissues using genes selected through the collection of methylation datasets, such as... Figure 14 As shown, all selected genes have been confirmed to have an area under the curve (AUC) value of 0.860 or higher, indicating diagnostic accuracy. Therefore, the selected genes can be used to diagnose hepatocellular carcinoma.

[0101] Example 5: Measurement of qMSP-based methylation of selected genes in liver cancer tissue Of the 13 selected genes, four genes—FAR1, PAK1, ATL1, and LIPE—were subjected to additional validation tests using cancer tissue. These genes exhibited significant differences in methylation levels between non-tumor and tumor tissues, and a higher average methylation level in tumor tissues. To confirm liver cancer-specific methylation of these four selected genes in cancer tissues, quantitative methylation-specific PCR (qMSP) was used to measure methylation differences between cancer and non-tumor tissues. For this purpose, genomic DNA was isolated from a pair of cancerous and adjacent normal tissues from a total of 15 liver cancer patients (five patients per stage from 2c to 4c), treated with bisulfite, and then the amplification and methylation levels of specific gene regions for each gene in FAR1, PAK1, ATL1, and LIPE were observed according to standard qMSP assays.

[0102] In addition, the ACTB gene was used as an internal control, which specifically binds to the region of the gene to be amplified that has been converted by bisulfite, and is independent of the methylation of the amplification value of the corresponding region.

[0103] The methylation level of bisulfite-converted DNA amplified by PCR is expressed as ΔCt+10, which is a value adjusted for the ACTB cycle threshold (Ct) used as an internal control. ΔCt+10 is defined as follows: ΔCt + 10 = (Ct value of the ACTB gene - Ct value of the gene to be tested) + 10 As shown in Figures 16A and 16B, the methylation of each of the genes FAR1, ATL1, PAK1, and LIPE was confirmed to show relatively high ΔCt + 10 values ​​in colorectal cancer tissues at all stages compared to adjacent normal tissues. Therefore, these four genes, FAR1, ATL1, PAK1, and LIP, are hypermethylated in hepatocellular carcinoma. This effectively demonstrates that the methylation of the selected genes FAR1, ATL1, PAK1, and LIPE is effective as a biomarker for diagnosis, particularly for the early diagnosis of hepatocellular carcinoma.

[0104] The results showed that the selected genes could even be used for the diagnosis of liver cancer.

[0105] Industrial applicability As described above, since hypermethylation of CpG sites selected from any one or more genes, including FAM110A, FAR1, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2, specifically occurs in liver cancer, liver cancer can be accurately and rapidly diagnosed at an early stage by using the compositions, kits, chips, or methods of the present invention.

Claims

1. Use of a kit in the preparation of a product for the specific and differentiated early diagnosis of liver cancer from samples obtained from patients suspected of having liver cancer. The patient samples mentioned above consist of cells, body fluids, or combinations thereof that appear clinically or morphologically normal. The kit contains a preparation for measuring the methylation level of CpG sites in FAR1 isolated from patient samples and optionally selected from one or more genes including FAM110A, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2; and The kit also contains at least one of the following: Primer pairs for amplifying fragments containing FAR1 and CpG sites optionally selected from one or more of the genes FAM110A, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2; and A nucleic acid chip having fixed probes capable of hybridizing with fragments containing CpG sites of FAR1 and optionally one or more genes selected from FAM110A, VIM, LDHB, LIPE, INAFM1, ATL1, CELF6, MTHFD2, PAK1, NXPE3, SLC25A36, and VANGL2; and The product is configured to specifically and differentially diagnose liver cancer at an early stage, distinguishing it from at least one of the following diseases: acute myeloid leukemia, adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrioid carcinoma, esophageal cancer, glioblastoma, head and neck cancer, chromophobe renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, large B-cell lymphoma, low-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, mesothelioma, ocular melanoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, sarcoma, gastric cancer, testicular cancer, thymoma, thyroid cancer, and uterine carcinosarcoma.

2. The use according to claim 1, wherein the CpG site is located between + / - 2000 bases (2 kb) from the transcription start site of the FAR1 gene and one or more optional genes.

3. The use according to claim 1, wherein the preparation for measuring the methylation level of CpG sites in the FAR1 gene and one or more optional genes comprises at least one selected from: Compounds capable of modifying unmethylated cytosine bases; Compounds capable of modifying methylated cytosine bases; Primers specific to the methylation sequence of the CpG site of the FAR1 gene and one or more optional genes; and Primers that are specific to unmethylated sequences.

4. The use according to claim 3, wherein the compound capable of modifying unmethylated cytosine bases is a bisulfite or a salt thereof, and the compound capable of modifying methylated cytosine bases is a Tet protein.

5. The use according to claim 1, wherein the sample is selected from tissues, cells, blood, plasma, serum, feces, and urine.

6. The use according to claim 1, wherein the preparation for measuring methylation levels comprises reagents for performing methods selected from: bisulfite-free detection methods, methylation-specific polymerase chain reaction, real-time methylation-specific polymerase chain reaction, PCR using methylation DNA-specific binding proteins, quantitative PCR, pyrosequencing, and bisulfite sequencing.

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