KR20130103110A - Method for detecting liver cancer using liver cancer specific hypermethylated cpg sequence - Google Patents

Abstract

PURPOSE: A method for detecting liver cancer using liver cancer-specific hypermethylated CpG sequence is provided to accurately and quickly diagnose liver cancer compared with a well-known method using serum which is a non-invasive sample. CONSTITUTION: A method for detecting liver cancer or liver cancer progression comprises the steps of: isolating DNA from a clinical sample; and detecting methylation at 47,909,729th-47,911,349th sequences of chromosome #1 which is a biomarker for liver cancer. A kit for diagnosing liver cancer contains a methylated site of 47,909,729th-47,911,349th sequences of chromosome #1. A nucleic acid chip for diagnosing liver cancer contains a probe which is hybridized with a fragment containing the methylated site. [Reference numerals] (AA) Analyze CpG microarray; (BB) Abutting normal opinion tissue: 3 sample; (CC) Liver cancer tissue: 3 sample; (DD) Refine methylation DNA by using MBD2bt; (EE) Amplify and Indicate fluorescence dye; (FF) Hybridization reaction; (GG) Bio marker

Description

Method for Detecting Liver Cancer Using Liver Cancer Specific Hypermethylated CpG Sequence {Method for Detecting Liver Cancer Using Liver Cancer Specific Hypermethylated CpG Sequence}

The present invention relates to a method for detecting liver cancer using a liver cancer-specific biomarker, and more particularly, to liver cancer comprising a liver cancer-specific hypermethylated CpG sequence or a liver cancer using a biomarker for diagnosis of its progression stage and its progression stage. It relates to a detection method.

Liver cancer is the third most common cancer / mortality rate in Korea, with men at 45/10/10 and 34/10/10, and women 12 / 100,000 and 8.8 / 100,000 a year. / Mortality (Yeun et al ., J. Gastroenterology & Hepatology, 24: 346, 2009).

Currently, AFP (alpha-feto protein) is used as a diagnostic marker for liver cancer, but due to its insufficient sensitivity and specificity, development of better diagnostic markers is required, and as a result of studies to develop diagnostic markers other than AFP, AFP-L3, Des-gamma carboxyprothrombin (DCP), alpha-L-Fucosidase, Glypican-3, Squamous cell carcinoma antigen (SCCA), bone protein protein (GP73), Hepatocyte growth factor (HGF), and transforming growth factor-beta1 (TGF-beta1) , Vascular endothelial cell growth factor (VEGF) has been developed and tested in the clinic (Gomaa et al., World J Gastro. 15: 1301, 2009).

As a method of diagnosing liver cancer, imaging methods such as ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and hepatic artery angiography (Angiography) are used in addition to measuring protein markers in blood. In addition, in the case of ultrasound, it is widely used as a primary imaging test for detecting the occurrence of liver cancer, and the sensitivity is greatly affected by the size of liver cancer. Large liver cancer tissues larger than 5 cm have a sensitivity of 75% or higher, whereas small liver cancers smaller than 1 cm have a sensitivity of about 42% (Gomaa et al., World J Gastro ., 15: 1301, 2009). Computed tomography (CT) is the most effective diagnostic method for liver cancer. Nearly 100% of liver cancers above 2 cm, 93% of 1-2 cm, and 60% of liver cancers of less than 1 cm can be diagnosed with sensitivity. (Gomaa et al., World J Gastro ., 15: 1301, 2009). However, due to the relatively high cost of the test, it is a burdensome test method for the routine screening test in the general public.

In the case of liver cancer, the size of the tumor at the time of diagnosis is closely related to the prognosis. The important thing for improving the survival rate of liver cancer patients is early detection of the tumor, and liver cancer is a relatively high frequency disease in Korea. Early detection through screening is important to reduce social costs associated with liver cancer. Therefore, liver cancer can be detected early with high sensitivity, and a diagnostic agent that can be screened at low cost is urgently needed.

The efficient cancer early diagnosis technology demanded by the market is highly accurate and should be able to diagnose non-invasive clinical samples (blood, urine, etc.) so that patients can be freely examined. As the diagnostic technology that best meets the requirements, it is expected to rapidly replace the existing clinical chemistry, clinical enzymatics and immunoassays to become the main field of the diagnostic test market within the next 10 years. In particular, molecular diagnosis technology using epigenetic-based nucleic acid biomarkers is expected to lead the future molecular diagnostics market.

Nucleic acid-based molecular diagnostic technology using methylated DNA biomarker is 1) high stability of sample based on nucleic acid, 2) early diagnosis to detect changes occurring early in cancer development, and 3) nucleic acid amplification technology. The detection sensitivity and specificity are high, and 4) several biomarkers can be analyzed, and thus the accuracy of clinical diagnosis is very high. Therefore, it is known as an optimal method for early diagnosis and recurrence monitoring of cancer.

Recently, methods for diagnosing cancer through DNA methylation measurement have been proposed. DNA methylation mainly occurs in the cytosine of the CpG island of the promoter region of a specific gene, In addition, methylation of the CpG islands of the promoter of the tumor suppressor gene is highly useful for cancer research. This is called methylation specific PCR (hereinafter referred to as "MSP"). ) Or automatic base analysis, and thus, attempts have been actively made for diagnosis and screening of cancer.

There is controversy as to whether methylation of the promoter CpG island directly induces carcinogenesis or causes secondary changes in carcinogenesis. However, in many cancers, tumor suppressor gene, DNA repair gene, Regulatory genes are hypermethylated and thus the expression of these genes is blocked. It is known that hypermethylation occurs in the promoter region of a specific gene at an early stage of cancer development.

Thus, the promoter methylation of tumor-associated genes is an important indicator of cancer, and it can be used in many aspects such as diagnosis and early diagnosis of cancer, prediction of carcinogenesis risk, prediction of cancer prognosis, follow-up after treatment and prediction of response to chemotherapy . Recently, attempts have been made to investigate the promoter methylation of tumor-associated genes in blood, sputum, saliva, stool, urine and the like and to use them in various cancer treatments (Esteller, M. et al., Cancer Res. , 59:67 , 1999: Sanchez-Cespedez, M. et al., Cancer Res ., 60: 892, 2000; Ahlquist, DA et al. , Gastroenterol. , 119: 1219, 2000).

Accordingly, the present inventors have made intensive efforts to develop a method for effectively detecting liver cancer. As a result, when the methylation degree is measured using a methylation-related chromosome sequence methylated in liver cancer as a biomarker, the progression of liver cancer and liver cancer is measured. It was confirmed that the present invention can be effectively detected, and the present invention was completed.

An object of the present invention is to provide a method for detecting liver cancer or liver cancer progression step using a methylation-related chromosomal sequence that is methylated specifically liver cancer as a biomarker.

Another object of the present invention to provide a liver cancer diagnostic kit and liver cancer diagnostic nucleic acid chip using a methylation-related chromosome sequence methylated specifically liver cancer as a biomarker.

The present invention comprises the steps of (a) separating the DNA from the clinical sample; And (b) detecting methylation of the 47,909,729-47,911,349th sequence of chromosome # 1, which is a liver cancer biomarker, in the separated DNA.

The present invention also provides a kit for diagnosing liver cancer containing a methylation site of the 47,909,729 to 47,911,349 sequence of chromosome # 1, which is a liver cancer biomarker.

The present invention also provides a nucleic acid chip for diagnosing liver cancer, comprising a fragment capable of hybridizing with a fragment comprising a methylation site of the 47,909,729 to 47,911,349th sequence of chromosome # 1, a liver cancer biomarker.

By using the biomarker of the present invention, early diagnosis of liver cancer can be performed more accurately and quickly than a conventional method, and serum, which is a non-invasive sample, can be used as a sample, so that liver cancer can be diagnosed more simply and accurately.

1 shows a process of discovering biomarkers by selecting hypermethylated sequences from liver cancer tissues through CpG microarray analysis of normal-tissue tissues adjacent to liver cancer tissues.
Figure 2 is a quantitative analysis of the degree of methylation of biomarkers in liver cancer cell line through pyro sequencing.
Figure 3 is a result of comparing the degree of methylation of biomarkers in normal tissue and liver cancer tissue.
Figure 4 is a measure of liver cancer diagnostic ability by performing ROC curve analysis. ( ^* criterion: cut-off; Sensitivity: sensitivity; Specificity: specificity).

In one aspect, the invention comprises the steps of (a) isolating DNA from clinical samples; And (b) detecting methylation of the 47,909,729 to 47,911,349th sequence of chromosome # 1, which is a liver cancer biomarker, in the separated DNA.

The liver cancer methylation biomarker according to the present invention was screened by the following method: (a) a protein in which DNA hypermethylation specifically binds a nucleotide sequence to a methylated sequence only in liver cancer tissue cells in liver cancer tissue and normal-tissue tissue Selectively selecting methylated DNA using (b) performing a CpG microarray experiment on the selectively selected methylated DNA; (c) sorting and selecting a list of hypermethyl sequences from the transformed tissue cells by comparing the methylation tendency of the transformed liver cancer tissue cells and the normal-tissue tissue cells of the junction region.

In the present invention, methylation detection of the 47,909,729 to 47,911,349 sequences of chromosome # 1, which is a liver cancer biomarker, may be performed at a DNA site having a nucleotide sequence of SEQ ID NO: 1.

In the present invention, the methylation detection is PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, DNA chip, pyro The method may be performed by a method selected from the group consisting of sequencing and bisulfite sequencing, and any method capable of detecting methylation of a nucleotide sequence may be used without limitation.

In the present invention, the clinical sample is detection method of liver cancer or liver cancer, characterized in that selected from the group consisting of tissue, cells, blood, plasma and urine suspected cancer or a diagnosis target.

In another aspect, the present invention relates to a kit for diagnosing liver cancer containing the methylation site of the 47,909,729 to 47,911,349 sequences of chromosome # 1, which is a biomarker.

In one aspect of the kit for use in the detection method of the present invention, there is provided a compartmentalized carrier means for holding a sample, a first container containing an agent that sensitively cleaves unmethylated cytosine, and a primer for amplifying CpG containing nucleic acid. One or more containers including a second container and a third container containing means for detecting the presence of cleaved or uncleaved nucleic acid. Primers used in accordance with the present invention include the sequences set forth in SEQ ID NOs: 3-6, functional combinations, and fragments thereof. A functional combination or fragment is used as a primer to detect whether methylation has occurred on the genome. The carrier means is suitable for containing one or more containers such as bottles, tubes, each container containing independent components used in the method of the invention. In the context of the present invention, one of ordinary skill in the art can readily dispense the required formulation in the container. For example, a container containing methylation sensitivity restriction enzyme may be configured as one container. One or more containers may contain primers that have homology with important nucleic acid locus. In addition, one or more of the containers may contain isoxisomers of methylation sensitive restriction enzymes.

In one aspect of the invention, using the kit, a method for detecting liver cancer comprises the steps of (1) separating DNA from clinical samples (2) treating the isolated DNA with methylation sensitive restriction enzyme (3) the Amplifying the DNA treated with methylation sensitive restriction enzyme using a primer capable of amplifying the liver cancer diagnostic biomarker of the present invention, and (4) determining the presence or absence of a liver cancer diagnostic biomarker in the amplified product. In addition, the biomarker fragment is present in the sample can be diagnosed as liver cancer or liver cancer progression sample, the kit is a hive under severe conditions and liver cancer diagnostic biomarker to confirm the presence of PCR amplification products using the included primers It may further contain fragments that can be redidated.

In addition, as a method for determining the presence or absence of the biomarker for diagnosing liver cancer, bisulfate sequencing, pyrosequencing, methylation-specific PCR, MethyLight, PCR using a methylated DNA binding protein, and a DNA chip may be used.

In another aspect, the present invention relates to a nucleic acid chip for diagnosing liver cancer comprising a fragment capable of hybridizing with a fragment comprising a methylation site of the 47,909,729 to 47,911,349 sequences of chromosome # 1, a liver cancer biomarker.

In PCR or DNA chip method using methylated DNA-specific binding protein, when a protein specifically binding to methylated DNA is mixed with DNA, only the methylated DNA can be selectively isolated because the protein specifically binds to the methylated DNA . Genomic DNA was mixed with methylated DNA-specific binding protein and only methylated DNA was selectively isolated. These isolated DNAs were amplified using a PCR primer corresponding to the promoter region and then methylated by agarose electrophoresis.

In addition, methylation can also be determined by quantitative PCR.Methylated DNA separated by methylated DNA-specific binding proteins can be labeled with a fluorescent dye and hybridized to DNA chips having complementary probes to measure methylation. Can be. Wherein the methylated DNA specific binding protein is not limited to MBD2bt.

In nucleic acid hybridization reactions used in the present invention, the conditions used to achieve stringent specific levels vary depending on the nature of the nucleic acid being hybridized. For example, hybridization conditions such as the length of the nucleic acid site to be hybridized, the degree of homology, the nucleotide sequence (for example, GC / AT composition ratio) and the nucleic acid type (for example, RNA or DNA) . An additional consideration is whether the nucleic acid is immobilized, for example, on a filter or the like.

Examples of very stringent conditions are as follows: 2X SSC / 0.1% SDS at room temperature (hybridization conditions); 0.2X SSC / 0.1% SDS at room temperature (low stringency conditions); 0.2X SSC / 0.1% SDS at 42 ° C. (conditions with moderate stringency); 0.1X SSC at 68 ° C. conditions with high stringency. The washing process can be carried out using one of these conditions, for example a condition with high stringency, or each of the above conditions, each 10-15 minutes in the order described above, all or all of the conditions described above. Some iterations can be done. However, as described above, the optimum conditions vary with the particular hybridization reaction involved and can be determined experimentally. In general, conditions of high stringency are used for hybridization of critical probes.

As a screening method of the methylated biomarker gene, not only liver cancer, but also various methylated genes can be found in various stages of dysplasia progressing to liver cancer, and the selected genes can be screened for liver cancer, risk assessment, prediction, disease identification, and disease stage It can also be used to select diagnostic and therapeutic targets.

Identifying genes that are methylated at liver cancer and at various stages of abnormality enables accurate and effective early diagnosis of liver cancer and can identify new targets for establishing and treating methylation lists using multiple genes. In addition, methylation data according to the present invention will be able to establish a more accurate liver cancer diagnosis system in conjunction with other non-methylated associated biomarker detection methods.

With the methods of the present invention, one can diagnose several stages or stages of liver cancer progression, including determining the methylation stage of one or more nucleic acid biomarkers obtained from a sample. By comparing the methylation step of nucleic acid isolated from the sample of each stage of liver cancer with the methylation step of one or more nucleic acids obtained from a sample having no cell proliferative abnormality of liver tissue, the specific stage of liver cancer of the sample can be identified. The step may be hypermethylation.

In one example of the invention, the nucleic acid may be methylated at the regulatory site of a gene. As another example, since methylation starts from the outside of the regulatory region of the gene and proceeds to the inside, detection of methylation at the outside of the regulatory region enables early diagnosis of genes involved in cellular transformation.

In another embodiment of the present invention, the methylation gene marker can be used for early diagnosis of cells likely to form liver cancer. When a gene identified to be methylated in cancer cells is methylated in cells that appear clinically or morphologically normal, the cells that appear to be normal are in progress of cancer. Therefore, liver cancer can be diagnosed early by confirming methylation of the liver cancer specific gene promoter region in cells that appear normal.

In one aspect of the invention, whether methylation of the liver biomarker methylation may include the following steps: (a) separating the DNA from the clinical sample; (b) treating bisulfite with the separated DNA; (c) amplifying using a primer capable of amplifying a fragment comprising a CpG island in the 47,909,729 to 47,911,349 sequences of chromosome # 1; And (d) performing pyro sequencing using the result amplified in step (c) to determine methylation.

In another embodiment of the present invention, by examining the methylation frequency of genes specifically methylated in liver cancer, and determining the methylation frequency of tissues with the possibility of liver cancer progression, the possibility of the development of the tissue into liver cancer can be evaluated.

As used herein, the term "cell transformation" refers to a process in which the characteristics of a cell differs from one form to another, such as from normal to abnormal, from non-positive to heterozygous, from undifferentiated to differentiated, It means to change. In addition, the transformation can be recognized by cell morphology, phenotype, biochemical properties, and the like.

In the present invention, "early identification" of cancer is to discover the possibility of cancer before metastasis, preferably before the morphological changes are observed in the specimen tissue or cells. In addition, "early identification" of cell transformation refers to the likelihood of transformation at an early stage before the cell is transformed into a form.

In the present invention, "hypermethylation" means methylation of CpG islands.

In the present invention, "sample" or "sample sample" means a wide range of samples, including all biological samples obtained from individuals, body fluids, cell lines, tissue culture and the like, depending on the kind of analysis performed. Methods for obtaining body fluids and tissue biopsies from mammals are commonly known. Preferred source is liver biopsy.

Screening of Methylation Regulated Biomarkers

In the present invention, biomarkers are screened for methylation when cells or tissues are transformed or when the cell's shape is changed to another form. Here, the term "transformed" cell means that the cell or tissue is changed into a different form such as a normal form, an abnormal form, a non-positive form, a non-differentiated form, or a differentiated form.

Therefore, in the present invention, the nucleotide sequence to be methylated in the transformation to liver cancer cells It was confirmed by a systematic method. In one embodiment of the method, genomic DNA was isolated from liver cancer tissue and adjacent normal-tissue tissue samples. The genomic DNA was reacted with MBD2bt protein that binds to methylated DNA and then amplified methylated DNA that binds to MBD2bt protein. Amplified DNA derived from liver cancer tissue and adjacent normal findings was labeled with Cy5, and internal control DNA was labeled with Cy3, and then hybridized to a human CpG microarray to express a large difference in methylation degree between adjacent normal tissue and liver cancer tissue. Were screened. Afterwards, various kinds of nucleotide sequences that were specifically methylated in liver cancer tissue were selected through various filtering steps.

In the present invention, the above To further confirm that the biomarkers were methylated, pyro sequencing was performed on liver cancer cell lines.

In other words, the whole genomic DNA was isolated from the hepatocarcinoma cell lines Hep3B (KCLB 30022), SNU449 (KCLB 00449) and HepG2 (KCLB 30026) to treat bisulfite, and then bisulfite-converted genomic DNA was specific for each marker. PCR was performed using a primer that can be amplified. Then, the amplified PCR product was subjected to pyrosequencing to measure the degree of methylation. As a result, all three liver cancer cell lines were identified as methylated, and finally, the hypermethyl CpG sequence present on chromosome # 1 was selected as the final biomarker.

Use of cancer cells for comparison with biomarker-normal cells for liver cancer

In this embodiment, "normal" cells refer to cells that do not exhibit abnormal cell morphology or changes in cytostatic properties. "Tumor" refers to a cancer cell, and "non-tumor" refers to a cell that is part of a diseased tissue but not a tumor.

In another use of the diagnostic kit of the present invention, the methylation stage of one or more nucleic acids isolated from a sample can be determined to early diagnose cell growth abnormalities of the liver tissue of the sample. The methylation step of the one or more nucleic acids may be compared with the methylation status of one or more nucleic acids isolated from a sample having no cell growth potential of liver tissue. The nucleic acid is preferably a CpG-containing nucleic acid such as a CpG island.

In another use of the diagnostic kit of the invention, one can diagnose abnormalities in cell growth of liver tissue of a sample comprising determining methylation of one or more nucleic acids isolated from the sample.

The term "soybean" means a property that is liable to suffer from the cell growth abnormality. A specimen with a lysate is a specimen that does not have any cell growth abnormality yet, but has an increased tendency to exist or to have a cell growth abnormality.

In another aspect, the invention provides contacting a sample comprising nucleic acid of a sample with an agent capable of determining the methylation state of the sample, and confirming cell growth of liver tissue of the sample comprising identifying methylation of one or more sites of one or more nucleic acids. It provides a method for diagnosing abnormalities. Wherein the methylation of at least one site of the at least one nucleic acid can be characterized as being different from the methylation step at the same site of the same nucleic acid of the sample that does not have a cell growthability abnormality.

The method of the invention comprises determining the methylation of at least one site of one or more nucleic acids isolated from a sample. Herein, the term "nucleic acid" or "nucleic acid sequence" means an oligonucleotide, a nucleotide, a polynucleotide or a DNA or RNA of a genomic or synthetic origin of a fragment, a single strand or a double strand thereof, a genomic origin or a synthetic DNA or RNA of origin, PNA (peptide nucleic acid), or a DNA quantity or RNA substance of natural or synthetic origin. It will be apparent to those skilled in the art that if the nucleic acid is RNA, it is replaced by ribonucleotides A, G, C and U, respectively, instead of deoxynucleotides A, G, C and T.

Any nucleic acid that can detect the presence of differently methylated CpG islands can be used. The CpG island is a CpG-rich region in the nucleic acid sequence.

Methylation

Any nucleic acid in purified or unpurified form may be used in the present invention, and any nucleic acid containing or suspected of containing a nucleic acid sequence containing a target site (eg, a CpG-containing nucleic acid) may be used. . A nucleic acid site that can be differentially methylated is a CpG island, which is a nucleic acid sequence having a high CpG density compared to other dinucleotide CpG nucleic acid sites. Doublet CpG is only 20% probable in vertebrate DNA when predicted by the ratio of G * C base pairs. At certain sites, the density of double CpG is ten times higher than at other sites in the genome. CpG islands have an average G * C ratio of about 60%, with the average DNA G * C ratio of 40%. CpG islands are typically about 1 to 2 kb in length and there are about 45,000 CpG islands in the human genome.

In many genes, CpG islands start upstream of the promoter and extend to the transcriptional site downstream. Methylation of CpG islands in promoters usually inhibits expression of genes. The CpG island may also surround the 5 'region of the gene coding region, as well as the 3' region of the gene coding region. Thus, CpG islands are found in several sites, including coding sequence upstream of the regulatory region comprising the promoter region, coding region (e.g., exon region), downstream of the coding region, such as the enhancer region and intron do.

Typically, the CpG-containing nucleic acid is DNA. However, the method of the present invention may apply for example a sample containing DNA or RNA containing DNA and mRNA, wherein the DNA or RNA may be single stranded or double stranded, or DNA-RNA hybrids. It may be characterized by the contained sample.

Nucleic acid mixtures may also be used. The specific nucleic acid sequence to be detected may be a fraction of a large molecule and the unique sequence from the beginning may exist in the form of a separate molecule constituting the entire nucleic acid sequence. The nucleic acid sequence need not be a nucleic acid present in a pure form, and the nucleic acid may be a small fraction in a complex mixture such as a whole human DNA. Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) used to measure the degree of methylation of the nucleic acid contained in the sample, or the nucleic acid contained in the sample used to detect the methylated CpG island, Can be extracted by various methods.

The nucleic acid may comprise a regulatory site, which is a DNA site that encodes information or regulates transcription of the nucleic acid. The regulatory site comprises at least one promoter. A "promoter" is the minimum sequence necessary to direct transcription and can regulate inducibility by cell type specific, tissue specific or external signal or agent in a promoter-dependent gene. The promoter is located at the 5 'or 3' site of the gene. Nucleic acid numbers of all or part of a promoter site can be applied to measure methylation of CpG island sites. Methylation of the target gene promoter proceeds from outside to inside. Therefore, the early stages of cell turnover can be detected by analyzing methylation outside of the promoter region.

The nucleic acid isolated from the sample is obtained by biological sample of the sample. To diagnose liver cancer or the stage of its progression, nucleic acids should be isolated from liver tissue with a scrap or biopsy. Such samples can be obtained by various medical procedures known in the art.

In the present invention, "hypermethylation" or "hypermethylation" refers to a state where the degree of methylation at a specific CpG position or a site connected to CpG of tumor cells is higher than that of normal cells.

Sample (sample)

The present invention describes early identification of liver cancer and utilizes liver cancer specific gene methylation. Methylation of liver cancer specific genes also occurred in tissues near the tumor site. Therefore, the early identification method of liver cancer can confirm the presence or absence of methylation of liver cancer-specific genes in all samples including liquid or solid tissue. The sample includes, but is not limited to, tissue, cells, urine, serum or plasma.

Individual genes and panels

The present invention can be used individually as a diagnostic or predictive marker, or a combination of two marker genes in the form of a panel display, wherein the two marker genes are weighted according to the presence and extent of the methylated genes together. And the level of likelihood of developing cancer. Such an algorithm belongs to the present invention.

Methylation detection method

Methylation specific PCR

When biosulfite is treated with genomic DNA, the cytosine in the 5'-CpG'-3 region is left as it is when it is methylated, and when it is unmethylated, it changes into uracil. Therefore, PCR primers corresponding to the sites where the 5'-CpG-3 'nucleotide sequence exists were prepared for the converted nucleotide sequence after bisulfite treatment. At this time, PCR primers corresponding to methylation and two types of primers corresponding to unmethylated primers were prepared. When genomic DNA is converted into bisulfite and then PCR is carried out using the above two types of primers, PCR products are produced by using primers corresponding to the methylated nucleotide sequence when methylated, and in the case of unmethylated PCR products are produced using primers corresponding to unmethylated sequences. The methylation can be qualitatively confirmed by agarose gel electrophoresis.

Real time methylation specific PCR (PCR)

Real-time methylation specific PCR is the conversion of methylation-specific PCR method to real-time measurement method. The PCR primer corresponding to the methylation of biosulfite treated with genomic DNA was designed and real-time PCR was performed using these primers . At this time, there are two methods of detection using a TaqMan probe complementary to the amplified base sequence, and two methods of detection using Sybergreen. Therefore, real-time methylation-specific PCR can quantitatively analyze only methylated DNA. At this time, a standard curve was prepared using an in vitro methylated DNA sample, and the methylation degree was quantitatively analyzed by amplifying a gene having no 5'-CpG-3 'sequence in the nucleotide sequence as a negative control for standardization.

Pyrosequencing

The pyro sequencing method is a method of converting the bisulfite sequencing method into quantitative real-time sequencing. As in bisulfite sequencing, genomic DNA was converted by bisulfite treatment, and then PCR primers corresponding to sites without the 5'-CpG-3 'sequence were prepared. After treating genomic DNA with bisulfite, it was amplified by the PCR primers, and real-time sequencing was performed using the sequencing primers. Quantitative analysis of cytosine and thymine at the 5'-CpG-3 'site indicated the methylation degree as the methylation index.

PCR or quantitative PCR using methylated DNA-specific binding protein and DNA chip

In PCR or DNA chip method using methylated DNA-specific binding protein, when a protein specifically binding to methylated DNA is mixed with DNA, only the methylated DNA can be selectively isolated because the protein specifically binds to the methylated DNA . Genomic DNA was mixed with methylated DNA-specific binding protein and only methylated DNA was selectively isolated. These isolated DNAs were amplified using a PCR primer corresponding to the promoter region and then methylated by agarose electrophoresis.

In addition, methylation can be measured using a quantitative PCR method. The methylated DNA separated by a methylated DNA-specific binding protein is labeled with a fluorescent dye and hybridized to a complementary probe-integrated DNA chip to measure methylation . Here, the methylated DNA-specific binding protein is not limited to MBD2bt.

Detection of differential methylation - methylation-sensitive restriction endonuclease

Detection of differential methylation can be accomplished by contacting the nucleic acid sample with a methylation sensitive restriction endonuclease that cleaves only the unmethylated CpG site and cleaving the unmethylated nucleic acid.

In a separate reaction, the sample was contacted with an isochisomer of a methylation-sensitive restriction endonuclease that cleaves both methylated and unmethylated CpG sites to cleave the methylated nucleic acid.

A specific primer was added to the nucleic acid sample and the nucleic acid was amplified by a conventional method. If the amplification product is present in the sample treated with the methylation-sensitive restriction endonuclease and the amplification product is not present in the sample treated with the methylation-sensitive restriction endonuclease isokisome that cleaves both methylated and unmethylated CpG sites , And methylation occurred at the analyzed nucleic acid site. However, even in samples that were treated with methylation-sensitive restriction endonuclease isokisomes that did not have an amplification product in the sample treated with the methylation-sensitive restriction endonuclease and cleaved both methylated and unmethylated CpG sites, Not present means that no methylation has occurred in the nucleic acid region analyzed.

Here, "methylation-sensitive restriction endonuclease" is a restriction enzyme that contains CG at the recognition site and has activity when C is methylated as compared to when C is not methylated (for example, Sma I). Non-limiting examples of methylation sensitive restriction endonuclease include Msp I, Hpa II, Bss HII, Bst UI and Not I. These enzymes can be used alone or in combination. Other methylation sensitive restriction endonucleotides include, but are not limited to Sac II and Eag I, for example.

Isokimers of methylation sensitive restriction endonuclease are limited endonucleases with the same recognition sites as methylation sensitive restriction endonucleases but cleave both methylated CGs and unmethylated CGs, for example, Msp I < / RTI >

The primers of the present invention are engineered to be "substantially" complementary to each strand of the locus to be amplified and comprise the appropriate G or C nucleotides, as described above. This means that the primer has sufficient complementarity to hybridize with the corresponding nucleic acid strand under the conditions of performing the polymerization reaction. The primers of the present invention are used in an amplification process, wherein the amplification process is an enzymatic sequencing reaction in which the target locus increases in exponential numbers through many reaction steps, such as, for example, PCR. Typically, one primer (antisense primer) has homology to the negative (-) strand of the locus and the other primer (sense primer) has homology to the positive (+) strand. Once the primer is annealed to the denatured nucleic acid, the chain is extended by enzymes and reagents such as DNA polymerase I (Klenow) and nucleotides, resulting in a new synthesis of the + and - strands containing the target locus sequence. When the newly synthesized target locus is also used as a template and the cycles of denaturation, primer annealing, and chain extension are repeated, exponential synthesis of the target locus sequence proceeds. The product of the continuous reaction is an independent double-stranded nucleic acid having a terminal end corresponding to the specific primer used in the reaction.

The amplification reaction is preferably a PCR commonly used in the art. However, alternative methods such as real-time PCR or linear amplification using isothermal enzymes can be used, and multiplex amplification reactions can also be used.

Detection of Differential Methylation-Bisulfite Sequencing Method

Another method of detecting a nucleic acid containing methylated CpG involves contacting a sample containing nucleic acid with an agent to modify unmethylated cytosine and amplifying the CpG-containing nucleic acid of the sample using a CpG-specific oligonucleotide primer . Here, the oligonucleotide primer may be characterized in that methylated nucleic acid is detected by distinguishing modified methylated and unmethylated nucleic acids. The amplification step is optional and desirable but not necessary. The method relies on PCR reactions to distinguish between modified (e. G., Chemically modified) methylated and unmethylated DNA. Such a method is disclosed in U.S. Patent 5,786,146, which is described in connection with bisulfite sequencing for the detection of methylated nucleic acids.

temperament

When the target nucleic acid region is amplified, the nucleic acid amplification product can be hybridized with a known gene probe immobilized on a solid support (substrate) in order to detect the presence of the nucleic acid sequence.

As used herein, the term "substrate" refers to a hybridization or enzymatic recognition site, such as a substance, structure, surface or material, abiotic, synthetic, inanimate, flat, spherical or specific binding, Or a large number of other recognition sites or a number of other recognition sites beyond a number of other molecular species composed of surfaces, structures or materials. Such substrates include, for example, semiconductors, (organic) synthetic metals, synthetic semiconductors, insulators and dopants; Metals, alloys, elements, compounds and minerals; Synthesized, degraded, etched, lithographic, printed and microfabricated slides, devices, structures and surfaces; Industrial, polymers, plastics, membranes, silicones, silicates, glass, metals and ceramics; But are not limited to, wood, paper, cardboard, cotton, wool, cloth, woven and nonwoven fibers, materials and fabrics.

Some types of membranes are known in the art to have an affinity for nucleic acid sequences. Specific, non-limiting examples of such membranes include nitrocellulose or polyvinyl chloride, diazotized paper, and membranes for gene expression detection such as commercially available membranes such as GENESCREEN ^™ , ZETAPROBE ^™ (Biorad) and NYTRAN ^™ . Beads, glasses, wafers and metal substrates. Methods for attaching nucleic acids to such objects are well known in the art. Alternatively, screening can be performed in a liquid phase.

Hybridization conditions

In nucleic acid hybridization reactions, the conditions used to achieve a certain level of stringency will vary depending on the nature of the nucleic acid being hybridized. For example, hybridization conditions such as the length of the nucleic acid site to be hybridized, the degree of homology, the nucleotide sequence (for example, GC / AT composition ratio) and the nucleic acid type (for example, RNA or DNA) . An additional consideration is whether the nucleic acid is immobilized, for example, on a filter or the like.

Examples of very stringent conditions include: 2X SSC / 0.1% SDS at room temperature (hybridization conditions); 0.2X SSC / 0.1% SDS at room temperature (low stringency conditions); 0.2X SSC / 0.1% SDS at 42 < 0 > C (conditions with normal stringency); 0.1X SSC at 68 ° C (conditions with high stringency). The cleaning process can be carried out using one of these conditions, for example a condition with high stringency, or the above conditions can be used respectively, and the conditions described above may be applied in whole or in part, Some can be done iteratively. However, as described above, optimal conditions vary depending on the particular hybridization reaction involved and can be determined through experimentation. Generally, conditions with high stringency are used for hybridization of critical probes.

Label

Important probes are detectably labeled and may be labeled, for example, as radioactive isotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. It is well known in the art to appropriately label such a probe, and can be carried out by a conventional method.

Kit

According to the present invention, there is provided a kit useful for detecting abnormality in cell growth of a specimen. The kit of the present invention comprises a first container containing a partitioned carrier means for containing a sample, an agent for sensitively cleaving unmethylated cytosine, a second container containing a primer for amplifying the CpG containing nucleic acid, and a second container containing a cleaved or uncut nucleic acid And at least one container comprising a third container containing means for detecting the presence.

Primers used according to the present invention may use the sequence, functional combinations and fragments thereof described in SEQ ID NOs: 2-3. A functional combination or fragment is used as a primer to detect whether methylation has occurred on the genome.

The carrier means is suitable for containing one or more containers such as bottles, tubes, each container containing independent components used in the method of the invention. In the context of the present invention, those skilled in the art will readily be able to dispense the necessary formulation in a container. For example, a container containing methylation sensitivity restriction enzyme may be configured as one container. One or more containers may contain primers that are homologous to the critical nucleic acid locus. In addition, the one or more containers may comprise an isokisome of a methylation sensitive restriction enzyme.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Screening for Specific Hypermethylated Sequences (Hymethyl Sequence, Hypermethylated Sequence) in Liver Cancer Paraffin Tissue Samples and Identification of Final Biomarkers

To select hypermethyl sequences from liver cancer, liver cancer tissues and adjacent normal-tissue tissue samples (Soncheon Hyang University College of Medicine, Pathology Laboratory) were selected as follows.

First, genomic DNA was isolated from three pairs of liver paraffin tissue samples (liver cancer tissue and adjacent normal-tissue tissue). 500 ng of the genomic DNA was fragmented into 300-400 bp by ultrasonic disruption (Vibra Cell, SONICS), and methylated DNA was selectively isolated from each tissue using Methylcapture ^™ (Genomictree, South Korea). Three normal-tissue tissues adjacent to liver cancer tissues were mixed to use a common reference as a common control. After amplification of the isolated methylated DNA using the GenomePlexComplete Whole Genome Amplification Kit (Sigma, USA), the common control DNA was labeled with Cy3-dUTP, and the amplified DNA derived from the normal-looking tissue adjacent to the amplified DNA derived from liver cancer tissue was Cy5-dUTP. After labeling and mixing, the hybridization was performed using a CpG microarray (Agilent, USA) in which probes for CpG present in 27,800 CpG islands present in the human genome were integrated. After the hybridization, a series of washing procedures were performed, followed by scanning using an Agilent scanner (Agilent, USA). The calculation of signal values from microarray images can be found in the Feature Extraction Program v. 9.5.3.1 (Agilent) was used to calculate the relative difference in signal intensity between adjacent normal-tissue and liver cancer tissue samples, and the difference in methylation level between adjacent normal and liver cancer tissues was analyzed using GeneSpringGX (Agilent). Large sequence regions were selected. Seven hypermethylated candidate targets were selected from liver cancer tissues through statistical techniques, and the candidate targets were subjected to various filtering steps. Finally, the hypermethyl CpG sequence present in chromosome # 1 (47,909,729 ~ 47,911,349nt) was finally obtained. Selection was made with biomarkers (FIG. 1).

Example 2: Determination of Methylation of Selected Biomarkers in Liver Cancer Cell Lines

Bisulfite pyro sequencing was performed to confirm the methylation status of the biomarkers selected in Example 1 in liver cancer cell lines.

In order to transform unmethylated cytosine into uracil using bisulfite, whole genomic DNA was isolated from liver cancer cell lines Hep3B (KCLB 30022), SNU449 (KCLB 00449) and HepG2 (KCLB 30026), and 200 ng of genome Bisulfite was treated with DNA using the EZ DNA methylation-Gold kit (Zymo Research, USA). Treatment of DNA with bisulfite leaves unmethylated cytosine modified with uracil and methylated cytosine remains unchanged. The bisulfite-treated DNA was eluted with 20 µl of sterile distilled water to perform pyrosequencing.

PCR and sequencing primers for performing pyro sequencing for the biomarkers were designed using the PSQ assay design program (Biotage, USA). PCR and sequencing primers are shown in Tables 1 and 2.

PCR primers and conditions primer Sequence (5'3 ') SEQ ID NO:
Amplicon size (bp)
PCR
forward TGGGTAGTGGTYGGGTAGGATAATTT 2 161
reverse Biotin-ACTAAAACTAAAAACCTCTATCCTATCT 3

Sequencing Primer Sequences of Methylated Biomarkers primer order SEQ ID NO: Sequencing TTGATTGGGTTTTTTGGA 4

20 ng of genomic DNA converted into bisulfite was amplified by PCR. PCR reaction solution (20 ng genomic DNA converted to bisulfite, 5 μl of 10X PCR buffer (Enzynomics, Korea), 5 units of Taq polymerase (Enzynomics, Korea), 4 μl of 2.5 mM dNTP (Solgent, Korea), 2 μl of PCR primer ( 10 pmole / μl)) was treated at 95 ° C. for 5 minutes, followed by a total of 45 times of 40 seconds at 95 ° C., 45 seconds at 60 ° C., and 40 seconds at 72 ° C., followed by reaction at 72 ° C. for 5 minutes. Amplification of the PCR product was confirmed by electrophoresis using 2.0% agarose gel.

The amplified PCR products were treated with PyroGold reagent (Biotage, USA) and pyrosequencing was performed using a PSQ96MA system (Biotage, USA). After the pyro sequencing, the degree of methylation was measured by calculating the methylation index. The methylation index was calculated by obtaining an average rate of cytosine binding at each CpG site.

As a result of quantitatively expressing the degree of methylation in the liver cancer cell line of the biomarker using the pyro sequencing method, it was confirmed that the methylation at high levels in all three cell lines (FIG. 2).

Example 3: Measurement of Hypermethylation of Biomarkers in Liver Cancer Tissues

As shown in Examples 1 and 2, in order to verify whether the biomarker is hypermethylated in liver cancer tissues and hypermethylated in liver cancer cell lines, it can be used as a biomarker for diagnosing liver cancer, and liver tissues of 25 normal persons, Methylation assay was performed on selected biomarkers using four normal buffy coats, one normal hepatocyte and lymphocyte, and 13 liver cancer tissue clinical specimens. The methylation assay was performed by the method described in Example 2 using the pyrosequencing method. As a result, the biomarkers showed a very high methylation level in the tissues of liver cancer patients compared to normal liver tissues (FIG. 3). In addition, methylation levels in normal buffy coats, hepatocytes and lymphocytes were also very low (FIG. 3A).

This fact can be applied to various diagnosis of liver cancer such as early diagnosis of liver cancer, recurrence, monitoring after drug treatment and monitoring of recurrence after surgery by measuring hypermethylation of selected bioker using serum, a noninvasive clinical sample. It can be said that there is.

ROC (receiver operating calculation) curve analysis was performed to determine the methylation index cut-off for the diagnosis of liver cancer patients, and to determine the sensitivity and specificity. As a result, it was confirmed that it is an excellent liver cancer diagnostic biomarker by showing the results of sensitivity 92.3%, specificity 100% (Fig. 4).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> GenomicTree Inc. <120> Method for Detecting Liver Cancer Using Liver Cancer Specific          Hypermethylated CpG Sequence <130> P12-B034 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1621 <212> DNA <213> homo sapiens <400> 1 aacttcgcct cctccagcgc cgccgcagcg cgcacttaat gaagttgaag gctcggtgag 60 cccgggtgaa gagggtttgg aatctgttga aaggggcgtt ttgtgacact gattggggcg 120 ggggcggggg cagtcgcgga ccagacaatg accgaccagg cgggttttcc tgcgcacagg 180 gtcaggcgaa taaaggcgcc gacgctgttt aaacgaagga ccttgtaaga gaaagggaga 240 aaagattttg tgtgtggagc gtgcctcgta aggtttcgtg cttaggagaa gttgggagga 300 ggcagctcgc ctagagtctt tacggaccga attcggagtt tatttcgaac actatgcatc 360 aagccaaaga aaagcggggc cagtttgggt ttgcgcctaa cttaattccg atacgcgcgt 420 caaaatgttg tgtaggctgg ggcctgggga ggcgttcagg agggccaacc aggaagatga 480 catccagccc acatttgtct ctgtggctga cgctctgagg tttggcgctc tggagaaacg 540 ttgaaagaaa actaaagatg ggcagtggtc gggcaggata actcatcctc ctaaagcgtt 600 tgtgagcaaa acaaatgttg attgggtttt ttggagcgga attactctgt tctttaaggt 660 cggcgcagac acgtaccagc agagaacctg tagacaggac agaggtttcc agctctagtt 720 tcgggaacgg atttttccgc gggctagtgg gcgcggcgcg gcgcagggcg gagcgggcac 780 cgcctccttc cgtagcggag cgagagctgc cgctcgaggc tgaggccgct ccggaggcct 840 ggaggcttcg gactgctgga ctagtgggga aggaaggcgg tttcctccgc agcgccccgg 900 tgctgcactc cgcaccgtca ccttctgggt tgtttctggc gctccctctg ctctcagcct 960 cgagtcctgg gtccctgcgg agcgctgtct tttcgctcca cgcggactct gagcggaagt 1020 cgctcgctgt ccgcgacttt gcatttctcc tgcagcaggg gtctctaccc ggtgccttcc 1080 tcccggcacg ctagcctcct cgccgaaatt tcgtcgtccc ggagtcggta accgagtccc 1140 aggctttact gccactccac tccctgctgg gttatttaag agatacgcgg cttccgaggg 1200 gttttcatca gaccccgcaa gtgcgctcgg ctgggaagga tgcgctccga tgccgctaca 1260 ggggttccgc gcgtttcacc gcgggaagcc cgggcattgg agatctgtgc tcagttctct 1320 aggtgttgga aagttgcctc aggctgcttt catctctggg gcatttctgt ctacgcaggc 1380 agaaggatct ttggaattgc agttgcatgg atcttagttc tggaaagggc gaatcattgg 1440 gggaacaaaa aattcaaatc cgtccttttg attcctaaaa atcactgcag acctgaaaag 1500 tgcatttcga gttatctgtg tagacactgc cggtgtgtgt gtggtggcgg agggtggggt 1560 aagggccgag ggtacaggag tggactttat tgttcacatt aactcaccgg tgcttacaga 1620 g 1621 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tgggtagtgg tygggtagga taattt 26 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 actaaaacta aaaacctcta tcctatct 28 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ttgattgggt tttttgga 18

Claims (9)

Method for detecting liver cancer or liver cancer progression comprising the following steps:
(a) separating DNA from a clinical sample; And
(b) detecting methylation of the 47,909,729 to 47,911,349 sequences of chromosome # 1, a liver cancer biomarker, in the separated DNA.
The method of claim 1, wherein methylation detection of the 47,909,729 to 47,911,349 sequences of chromosome # 1, which is a liver cancer biomarker, is performed at a DNA region having a nucleotide sequence of SEQ ID NO: 1.
The method of claim 1, wherein the methylation detection is performed by PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, DNA chip, pi A method for detecting liver cancer or liver cancer progression, characterized in that performed by a method selected from the group consisting of rosequencing and bisulfite sequencing.
The method of claim 1, wherein the clinical sample is selected from the group consisting of tissue, cells, blood, plasma and urine suspected of cancer or a diagnosis target.
Liver cancer diagnostic kit containing a methylation site of the 47,909,729 ~ 47,911,349 sequence of chromosome # 1, a liver cancer biomarker.
The kit for diagnosing liver cancer according to claim 5, wherein the methylation site of the 47,909,729 to 47,911,349 sequence of chromosome # 1, which is a liver cancer biomarker, has a nucleotide sequence of SEQ ID NO: 1.
A nucleic acid chip for liver cancer diagnosis, comprising a fragment comprising a methylation site of the 47,909,729 to 47,911,349 sequences of chromosome # 1, which is a liver cancer biomarker, and a probe capable of hybridization.
The nucleic acid chip of claim 7, wherein the methylation site of the 47,909,729 to 47,911,349 sequence of chromosome # 1, which is a liver cancer biomarker, has a nucleotide sequence of SEQ ID NO: 1.
The nucleic acid chip for diagnosing liver cancer according to claim 7, wherein the hybridization is performed under stringent conditions.

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