KR20220041402A - Novel biomarker for diagnosing liver cancer - Google Patents

Abstract

The present invention provides a biomarker composition for diagnosing liver cancer, comprising at least one selected from a group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1. The biomarkers according to the present invention exhibit excellent effects in diagnosing liver cancer, and can be usefully used in classifying liver cancer patients, selecting a therapeutic agent accordingly, and establishing a treatment plan.

Description

New biomarker composition for liver cancer diagnosis {NOVEL BIOMARKER FOR DIAGNOSING LIVER CANCER}

The present invention relates to a novel biomarker composition for diagnosing liver cancer, and a method for diagnosing liver cancer using the same.

Cancer has a high mortality rate worldwide and is the second most common cause of death in Western societies after cardiovascular disease. In particular, with the aging of the population, lung cancer is increasing due to an increase in the smoking population and air pollution. The consumption of a high-fat diet has become common due to the westernization of diet, and liver cancer and colorectal cancer are caused by the rapid increase in environmental pollutants and the increase in the amount of alcohol consumed. , breast cancer, prostate cancer, etc. are continuously increasing. In this situation, there is an urgent need to create anticancer substances that can contribute to the promotion of human health, the improvement of the quality of healthy life, and the promotion of human health by enabling the early prevention and treatment of cancer.

Among them, liver cancer is the most common high-risk Korean cancer, accounting for more than 22% of all cancer deaths in Korea (as of 2015). In addition, the treatment cost per patient is about 66.22 million won, which is the most expensive cancer among Korean cancers, and it is a major disease that increases the social and economic medical burden. Bayer's Nexavar is currently the only treatment for liver cancer, and it is growing at an average annual rate of more than 10%, forming a market worth 1.2 trillion won per year.

Diagnosis of liver cancer was mainly carried out as a surveillance test for clearly high-risk groups, but less than 50% of patients diagnosed with liver cancer in Korea are detected by screening. To date, biomarkers using AFP and PIVKA II proteins have been developed for liver cancer diagnosis, but the development of high-performance biomarkers and diagnostic systems that can predict the treatment effect and prognosis for each liver cancer patient is sluggish. In addition, a large-scale genome/transcriptome/epigenome analysis for cancer was recently conducted in a large-scale oncogenome project such as TCGA (The Cancer Genome Atlas) to report a number of genomic/transcriptome/epigene mutations and suggest new molecular subtypes. However, biomarkers related to the prognosis of liver cancer patients have not yet been systematically developed.

On the other hand, transcription by RNA polymerase occurs in about 90% of the human genome, and most RNAs are noncoding RNAs that do not encode proteins. These noncoding RNAs are classified according to their length and function, and studies on the functions of transfer RNA, ribosomal RNA, miRNA, siRNA, and long noncoding RNA (lncRNA) are being actively conducted. Recently, as a type of novel noncoding RNA, the existence of an enhancer RNA (eRNA) made from an activated enhancer has been revealed. Depending on the length and production method, eRNA is divided into 1D (unidirectional) eRNA and 2D (bidirectional) eRNA. It is known that 5' capping occurs but not splicing or polyadenylation, unlike lncRNA, which has been actively studied recently.

Therefore, there is a need to understand the pathogenesis of cancer by discovering Korean liver cancer-specific enhancers and eRNAs based on liver cancer cell lines, patient-derived tissues and organoids, and to suggest new targets for liver cancer diagnosis and treatment. Moreover, efforts are needed to develop a liver cancer-specific prediction system by elucidating the regulatory mechanisms of novel enhancers and eRNAs.

Korean Patent Publication No. 10-2019-0024379 Korean Patent Publication No. 10-2019-0002874

Accordingly, the present inventors performed integrated transcriptome analysis using liver cancer cell lines, and ChIP-sequencing was performed on histone H3, H3K4me1 and H3K27ac to discover activated enhancers in liver cancer cell lines. seq was performed to perform integration analysis with enhancer peaks obtained from histone ChIP-seq.

Accordingly, activated enhancers were discovered from liver cancer cell lines, and THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, AC104046.1 were analyzed through the data of The Cancer Genome Atlas (TCGA). As a result of the analysis, it was found to be increased in liver cancer patients compared to normal people.

Based on these experimental results, it is confirmed that THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, AC104046.1 can be used as specific biomarkers for liver cancer. Confirmed and completed the present invention.

In particular, when using the biomarkers according to the present invention, the excellent effect as a marker is recognized in that it was confirmed that both ChIP-seq analysis and TCGA showed the same results.

One object of the present invention is to diagnose liver cancer comprising any one or more biomarkers selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 To provide a biomarker composition.

Another object of the present invention is the expression of any one or more eRNAs (biomarkers) selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 It is to provide a composition for diagnosis of liver cancer, including an agent for measuring the level.

THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 for diagnosis of liver cancer, including an agent for measuring the expression level of any one or more eRNAs selected from the group consisting of to provide a kit.

Another object of the present invention is (a) any one selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 from an isolated biological sample measuring one or more eRNA expression levels;

(b) any one or more selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 of a normal control sample for comparing the measured expression level comparing the expression level; and

(c) the expression level of any one or more selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 of the biological sample of a normal control in contrast THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and when higher than the expression level of any one or more selected from the group consisting of AC104046.1 liver cancer, including the step of determining as liver cancer It is to provide a method for providing information for diagnosis.

In order to achieve the above object, the present invention provides liver cancer comprising at least one biomarker selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 It provides a biomarker composition for diagnosis of

The one or more eRNAs may be used independently or in combination as a biomarker for diagnosis of liver cancer.

Liver cancer refers to a tumor in which hepatocytes lose their inherent function due to continuous various stimuli, transform into cancer cells, and spread to nearby or distant places while achieving constant self-renewal. metastatic cancer that has spread to Primary liver cancer includes hepatocellular carcinoma caused by abnormalities in hepatocytes, cholangiocarcinoma and angiosarcoma caused by abnormalities in bile duct cells, and more than 90% is hepatocellular carcinoma (HCC).

Liver cancer according to the present application is a primary malignant tumor that occurs in a tissue itself, and is hepatocellular carcinoma. Hepatocellular carcinoma is frequent in high-risk patients with risk factors such as alcohol abuse, viral hepatitis and cirrhosis or metabolic liver disease including cirrhosis. Hepatocellular carcinoma does not have a fibrous stroma, causing hemorrhage and cell necrosis, vascular infiltration into the portal vein system, and, in severe cases, can lead to liver rupture and intraperitoneal blood effusion.

The diagnosis and stage of cancer generally determines the stage of cancer progression according to the type of cancer cell, the degree of differentiation, the degree of metastasis, etc. through a biopsy of a tissue obtained through surgery or a biopsy. That is, the treatment and prognosis of general solid cancer are determined by the stage of TNM.

However, in the case of liver cancer, it may be difficult to select a treatment method or predict a prognosis based on the TNM stage alone. This is because chronic liver disease develops concurrently in most liver cancer patients, and treatment options are limited only with advanced liver disease independent of cancer.

Therefore, when the marker according to the present invention is used, the onset of liver cancer can be monitored and early diagnosis can be made. Diagnosis in the present invention means confirming the presence or characteristics of a pathological condition. For the purpose of the present invention, diagnosis is to determine whether or not liver cancer occurs, the prognosis of liver cancer, the possibility of progression to liver cancer, and the like. For example, it refers to determining the disease name, and may include the disease name, disease state, stage, etiology, presence or absence of complications, prognosis, and recurrence of liver cancer.

In the present invention, the biomarker is a substance that can distinguish liver cancer from normal hepatocytes and the like, and is a polypeptide or nucleic acid (eg, eRNA, mRNA, etc.), lipid, glycolipid, glycoprotein or sugar (monosaccharide) showing an increase or decrease in liver cancer. , disaccharides, oligosaccharides, etc.) and the like). For the purpose of the present invention, biomarkers for diagnosing liver cancer are THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, AC104046 that show a specific high level of expression in liver cancer compared to normal liver cells. It is any one or more eRNAs selected from the group consisting of .1.

In the present invention, enhancer RNA (eRNA) is known to be made from an activated enhancer as a type of noncoding RNA.

In the present invention, THUMPD3-AS1 (THUMPD3 antisense RNA 1) (ENSG00000206573) information is registered as GeneID: 440944 in the National Center for Biotechnology Information (NCBI).

In the present invention, MIR4435-2HG (MIR4435-2 host gene) (ENSG00000172965) information is registered as GeneID: 541471 in NCBI (National Center for Biotechnology Information).

In the present invention, PLEKHA8P1 (pleckstrin homology domain containing A8 pseudogene 1) (ENSG00000134297) information is registered as GeneID: 51054 in the National Center for Biotechnology Information (NCBI).

In the present invention, LINC00511 (long intergenic non-protein coding RNA 511) (ENSG00000227036) information is registered as GeneID: 400619 in the National Center for Biotechnology Information (NCBI).

In the present invention, (ENSG00000182648) information of LINC01006 (long intergenic non-protein coding RNA 1006) is registered as GeneID: 100506380 in NCBI (National Center for Biotechnology Information).

In the present invention, information on HOXA-AS2 (HOXA cluster antisense RNA 2) (ENSG00000253552) is registered as GeneID: 285943 in the National Center for Biotechnology Information (NCBI).

In the present invention, the information of AC019080.1 (ENSG00000213963) is registered as GeneID: 100130691 in the National Center for Biotechnology Information (NCBI), and is one of the ailas names of LOC100130691.

AC104046.1 (ENSG00000259630) in the present invention is known as a Pseudogene of Fatty Acid Binding Protein 5 (Psoriasis-Associated) (FABP5).

According to one embodiment of the present invention, THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, AC104046.1 were analyzed through data liver cancer samples from The Cancer Genome Atlas (TCGA). As a result, it was found to be increased in liver cancer patients compared to normal people.

Based on these experimental results, it is confirmed that THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, AC104046.1 can be used as specific biomarkers for liver cancer. Confirmed and completed the present invention.

In the present invention, the biomarker composition may include alone or a combination of two or more biomarkers without limitation.

The present invention also includes an agent for measuring the expression level of any one or more eRNAs selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 It provides a composition for diagnosis of liver cancer.

The expression level of any one or more biomarkers selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 can be determined by checking the expression level of eRNA. there is.

In the present invention, "eRNA expression level measurement" is a process of confirming the presence and expression level of the eRNA in a biological sample for diagnosing liver cancer, and can be known by measuring the amount of eRNA. Analysis methods for this include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA; RNase protection assay), Northern blotting (Northern blotting). blotting) and a DNA chip, but is not limited thereto.

The agent for measuring the eRNA level is preferably a pair of primers or a probe, and since the nucleic acid sequence of the genes of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, AC104046.1 is known, those skilled in the art can design a primer or probe that specifically amplifies a specific region of these genes based on the sequence.

In the present invention, the primer is a nucleic acid sequence having a short free 3' hydroxyl group, which can form a complementary template and base pair, and serves as a starting point for template strand copying. It refers to a short nucleic acid sequence. Primers can initiate synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) in an appropriate buffer and temperature.

In the present invention, PCR amplification is performed using sense and antisense primers of one or more genes selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1. Thus, liver cancer can be diagnosed through the production of a desired product. PCR conditions, the length of the sense and antisense primers can be modified based on what is known in the art.

In the present invention, the probe refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as long as possible to achieve specific binding to eRNA, and is labeled to confirm the presence or absence of a specific eRNA. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In the present invention, hybridization is performed using a probe complementary to one or more polynucleotides selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1. , it is possible to diagnose liver cancer through hybridization. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, “encapsulation”, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (eg, methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

In the present invention, the composition for diagnosis of liver cancer may include, without limitation, an agent for measuring the expression level of eRNA for single or a combination of two or more biomarkers.

In addition, if necessary, any known method capable of confirming the amount of the marker can be used without limitation, so for the analysis of multiple markers, that is, a large amount of eRNA, Chromatin Immunoprecipitation (ChIP)-Seq, ChIP-qPCR, ATAC (Assay) In the group consisting of for Transposase-Accessible Chromatin)-Seq, DNase-seq, FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)-seq, CLIP (Cross-linking immunoprecipitation)-seq, RIP (RNA immunoprecipitation)-seq and luciferase assay Any one or more selected may be used, but is not limited thereto.

The present invention also comprises an agent for measuring the expression level of any one or more eRNAs selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 , a kit for diagnosing liver cancer.

The kit of the present invention includes any one or more biomarkers selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1, which are biomarkers for liver cancer diagnosis, That is, it can be detected by checking the expression level of eRNA. The diagnostic kit of the present invention may include primers and probes for measuring the expression level of liver cancer biomarkers, as well as one or more other component compositions, solutions or devices suitable for the analysis method.

Specifically, the kit for measuring the expression level of any one or more eRNAs selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1 and AC104046.1 is RT-PCR It may be a kit comprising the necessary elements necessary to perform it. The RT-PCR kit includes, in addition to each primer pair specific for a biomarker gene, a test tube or other appropriate container, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq- enzymes such as polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPCwater, sterile water, and the like. It may also include a pair of primers specific for a gene used as a quantitative control. Also preferably, the kit of the present invention may be a diagnostic kit including essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA corresponding to a gene or fragment thereof is attached as a probe, and reagents, agents, enzymes, and the like for producing a fluorescently-labeled probe. In addition, the substrate may include cDNA corresponding to a quantitative control gene or a fragment thereof. For example, the reagent for nucleic acid amplification may be a polymerase, dNTP, buffer, nucleic acid, coenzyme, fluorescent material, or a combination thereof. The polymerase is, for example, Taq polymerase. The kit may further include a reagent for nucleic acid isolation. The reagent for nucleic acid isolation may include a cell lysis solution. The cell lysis solution may include a salt, a surfactant, a metal ion, a sugar, a reducing agent (eg, DTT), or a combination thereof.

In the present invention, the kit for diagnosing liver cancer may include, without limitation, an agent for measuring the expression level of eRNA for single or a combination of two or more biomarkers as in the salpin composition above, preferred examples are as described above salpin .

The present invention measures the expression level of any one or more selected from the group consisting of (a) THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 from an isolated biological sample to do;

(b) any one or more selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 of a normal control sample for comparing the measured expression level comparing the expression level; and

(c) the expression level of any one or more selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 of the biological sample of a normal control in contrast THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and when higher than the expression level of any one or more selected from the group consisting of AC104046.1 liver cancer, including the step of determining as liver cancer A method for providing information for diagnosis is provided.

The expression level of steps (a) and (b) may be detected at the eRNA level, and eRNA isolation from a biological sample may be performed using a known process.

In the present invention, the term "sample" includes samples such as cells, tissues, whole blood, serum, plasma, cerebrospinal fluid, saliva, sputum or urine, and is preferably a liver tissue sample.

Through the measurement of the expression level, it is possible to diagnose the onset and progression of liver cancer in a suspected liver cancer patient by comparing the eRNA expression level in the normal control with the eRNA expression level in the liver cancer suspected patient.

That is, after measuring the expression level of the biomarker of the present invention from cells presumed to be liver cancer, and measuring the expression level of the marker of the present invention from normal cells, and comparing both, the expression level of the marker of the present invention is that of normal cells. If it is expressed more than that, it is presumed to be liver cancer, and accordingly, it can be predicted as liver cancer.

Analytical methods for measuring eRNA levels include, but are not limited to, a reverse transcriptase polymerase reaction, a competitive reverse transcriptase polymerase reaction, a real-time reverse transcriptase polymerase reaction, an RNase protection assay, Northern blotting, and a DNA chip. Through the above detection methods, the eRNA expression level in the normal control group and the eRNA expression level in patients with suspected liver cancer can be compared, and the occurrence of a suspected liver cancer patient can be determined by determining whether the expression level of the liver cancer biomarker gene to eRNA is significantly increased. can be diagnosed

The eRNA expression level is preferably measured using a reverse transcriptase polymerase reaction method or a DNA chip using a primer specific for a gene used as a liver cancer biomarker.

The reverse transcriptase polymerase reaction is electrophoresed after the reaction to confirm the band pattern and the thickness of the band, thereby confirming the presence and level of eRNA expression of a gene used as a liver cancer biomarker. can be diagnosed

The DNA chip uses a DNA chip in which a nucleic acid corresponding to the liver cancer biomarker gene or a fragment thereof is attached to a glass-like substrate at a high density, isolates eRNA from a sample, and a cDNA probe labeled with a fluorescent material at the end or inside. is prepared, hybridized to a DNA chip, and then the occurrence of liver cancer can be read.

In the present invention, the method of providing information for the diagnosis of liver cancer may include, without limitation, measurement of the expression level for a combination of two or more biomarkers as in the above salpin composition, and a preferred example is as described above salpin.

The present invention measures the expression level of any one or more selected from the group consisting of (a) THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 from an isolated biological sample to do;

(b) any one or more selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 of a normal control sample for comparing the measured expression level comparing the expression level; and

(c) the expression level of any one or more selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 of the biological sample of a normal control in contrast THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and determining the liver cancer when higher than the expression level of any one or more selected from the group consisting of AC104046.1; and

(d) provides a method for treating liver cancer comprising administering a pharmaceutically effective amount of an anticancer agent to the patient determined to be liver cancer.

The pharmaceutically effective amount means an amount sufficient to inhibit or alleviate an increase in vascular permeability at a reasonable benefit/risk ratio applicable to medical use, and the effective dose level includes the subject type and severity, age, sex, drug activity, drug It can be determined according to factors including sensitivity to, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

The administration refers to introducing an anticancer agent to a subject in any suitable method, and the administration route may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue.

Any one or more biomarkers selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 according to the present invention show an excellent effect in diagnosing liver cancer , it can be usefully used to classify liver cancer patients, select a therapeutic agent accordingly, and establish a treatment plan.

1 shows a schematic diagram of the entire process for liver cancer-specific enhancer and eRNA discovery.
2 shows the results of analyzing eRNAs highly expressed in liver cancer compared to normal controls based on TCGA (The Cancer Genome Atlas) data.

Hereinafter, preferred examples and formulation examples are presented to help the understanding of the present invention. However, the following Examples and Formulation Examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited by the Examples or Formulation Examples.

< Experimental example 1> ChIP - seq (Chromatin Immunoprecipitation -sequencing) analysis

A total of 8 liver cancer (HCC) cell lines (FT 3-7, Huh 7, Huh 7-5, PCLC PRF 5, SNU 182, SNU 387, SNU 449, HepG2) were used for liver cancer-specific enhancer and eRNA discovery.

HCC cell lines were fixed with 1% paraformaldehyde and the reaction was stopped by adding glycine. The pellet was dissolved in lysis buffer (5 mM PIPES pH 8.0, 85 nM KCl, 0.5% NP40, 1X protease inhibitor (Roche)) and sonication buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA pH 8.0, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100) was sonicated (diagenode) in milliTUBE or microTUBE for 60 min until most fragment sizes were in the range of 200 to 300 bp. For each immunoprecipitation, antibody was added and spun at room temperature for 2 h to bind to beads (Dynabeads™ Protein A, Invitrogen). The antibodies used were H3 (2 μg per immunoprecipitation, Abcam ab1791), H3K27ac (2 μg per immunoprecipitation, Abcam ab4729) and H3K4me1 (2 μg per immunoprecipitation, Abcam ab8895). For the control library, immunoprecipitation with 1 μg of non-specific IgG mouse antibody was used. The blocked antibody-conjugated beads were placed on a magnet, the supernatant was removed, the sonicated lysate was added to the beads, and then incubated for 3-4 hours at 4 °C on a rotator. After washing the beads, the tags were reacted (Nextra® DNA Library Prep Kit, illumina). Wash the beads and elute overnight at 65 °C with 1X TE buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA) containing 2.5 μl of Proteinase K (Roche) and 1.5 ul of 10% SDS to cross-formaldehyde. The linking was reversed. Finally, DNA was purified with AMPure XP beads and each library was amplified (Nextra® DNA Library Prep Kit, Nextra® Index Kit, illumina). The amplified library was purified using AMPure XP beads and then size was selected using AMPure XP beads to recover a library with a fragment length of 200-400 bp. RNA library sequencing was performed on an Illumina HiSeq2000 platform.

< Experimental example 2> ChIP - seq and Total RNA- seq analyze

ChIP-seq was performed for H3K4 monomethylation (H3K4me1) and H3K27 acetylation to discover activated enhancers. In addition, total RNA-seq was performed by extracting total RNA from the 8 hepatocarcinoma cell lines to discover eRNA expressed from the activated enhancer.

Specifically, for chip seq, read quality check was performed with 'FastQC', and then read filtering was performed with 'Trimmomatic'. After alignment with ‘Bowtie2’, a BAM file with non-uniquely aligned reads and potential PCR duplicates removed was created. After that, peak calling was performed using ‘MACS14’ and peak annotation was performed with ‘HOMER’.

In addition, for RNA seq, After the read quality check was performed with 'FastQC', read filtering was performed with 'Trimmomatic'. Total RNA-seq reads were aligned to the reference genome using 'Tophat'. As the reference genome, GRCh38 of Ensembl was used. After that, 'Cufflinks' was performed, and the expression level of the gene was calculated using FPKM.

< Experimental example 3> liver cancer specific enhancer and eRNA excavation

For the discovery of the specifically activated enhancer and eRNA expressed therefrom in hepatocellular carcinoma cell lines, H3K27 acetylation peaks, which are known to be high in the activated enhancer, were selected on the entire genome. In order to discover only the activated enhancer, H3K27 acetylation peaks appearing in the gene promoter or inside were excluded. Next, the peaks of H3K4 monometylation (H3K4me1), known as a marker for enhancer, were selected from the entire genome, and finally, an activated enhancer site in which H3K27 acetylation and H3K4 monomethylation were simultaneously high was selected.

In addition, in order to discover the eRNA expressed in the activated enhancer, total RNA-seq data and integrated analysis were performed. A series of analysis schematic diagrams related thereto is shown in FIG. 1 .

< Experimental example 4> TCGA (The Cancer Genome Atlas) Validation using data

The expression patterns of the selected eRNAs were analyzed using RNA-seq data produced from 50 samples of Jeongsanin liver cancer and 50 samples of liver cancer patients.

<Experiment result>

One. in liver cancer cell lines expressed eRNA excavation and analysis

The activated enhancer and eRNA expressed therefrom were analyzed using a total of 8 hepatocellular carcinoma cell lines. Specifically, a total of 45 eRNAs were selected using 4 hepatocellular carcinoma cell lines (F-3-7, Huh-7-5, SNU182, PLC-PRF-5) with similar gene expression patterns among 8 hepatocellular carcinoma cell lines. Specific analysis results are shown in Table 1 below.

[Table 1]

As can be seen in Table 1, it was confirmed that a total of 45 eRNAs were expressed in hepatocellular carcinoma cell lines. The eRNAs identified above included THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1.

2. TCGA Validation using data

Expression patterns of 45 eRNAs discovered using TCGA data were confirmed. A total of 11 eRNAs had increased expression in liver cancer tissues (50 cases) compared to normal liver tissues (50 cases). 11 biomarker eRNAs discovered in liver cancer cell lines through this result are shown in FIG. 2 and Table 2.

Increased expression in liver cancer eRNA Increased expression in liver cancer eRNA THUMPD3-AS1 MIR4435-2HG PLEKHA8P1 LINC00511 LINC01006 HOXA-AS2 SAP30L-AS1 AC019080.1 TRAF3IP2-AS1 AC104046.1 LINC01572

As can be seen in Table 2 and FIG. 2, it was confirmed that the expression of a total of 11 biomarker eRNAs was increased in liver cancer tissues compared to normal liver tissues.

Based on the above results, the expression of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 according to the present invention is greatly increased in liver cancer tissue, This showed a significant difference when compared with the normal control group.

These results show that the above-mentioned biomarkers can be usefully used for the diagnosis of liver cancer.

Claims (8)

THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 A composition for diagnosis of liver cancer, comprising an agent for measuring the expression level of any one or more eRNAs selected from the group consisting of . The composition for diagnosis of liver cancer according to claim 1, wherein the agent for measuring the expression level of the eRNA comprises a primer that specifically binds to the biomarker. According to claim 1, wherein the measurement of the expression level of eRNA is RT-PCR, competitive RT-PCR (Competitive RT-PCR), real-time RT-PCR (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay) ), Northern blotting (Northern blotting) and by any one selected from the group consisting of a DNA chip, a composition for diagnosis of liver cancer. The method of claim 1, wherein the measurement of the expression level of eRNA is any one selected from the group consisting of ChIP-Seq, ChIP-qPCR, ATAC-Seq, DNase-seq, FAIRE-seq, CLIP-seq, RIP-seq and luciferase assay At least one composition for diagnosis of liver cancer. A kit for diagnosing liver cancer, comprising the composition of any one of claims 1 to 4. (a) measuring the expression level of any one or more selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 from the isolated biological sample;
(b) any one or more selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 of a normal control sample for comparing the measured expression level comparing the expression level; and
(c) the expression level of any one or more selected from the group consisting of THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and AC104046.1 of the biological sample as a contrasting normal control THUMPD3-AS1, MIR4435-2HG, PLEKHA8P1, LINC00511, LINC01006, HOXA-AS2, AC019080.1, and when higher than the expression level of any one or more selected from the group consisting of AC104046.1 liver cancer, including the step of determining as liver cancer How to provide information for diagnosis. The method of claim 6, wherein the expression level measurement in step (a) is to measure the expression level of eRNA, a method for providing information for diagnosing liver cancer. According to claim 6, wherein the measurement of the expression level in step (a) is RT-PCR, competitive RT-PCR (Competitive RT-PCR), real-time RT-PCR (Real-time RT-PCR), RNase protection assay (RPA) ; RNase protection assay), Northern blotting, and a method of providing information for liver cancer diagnosis by any one selected from the group consisting of a DNA chip.

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