KR102216943B1 - Biomarker for diagnosing liver cancer - Google Patents

Abstract

The present invention provides a biomarker composition for liver cancer diagnosis, which includes one or more selected from a group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS. According to the present invention, the biomarkers show 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

Biomarker composition for diagnosis of liver cancer {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 most common cause of death after cardiovascular disease in Western society. In particular, lung cancer is increasing due to an increase in the smoking population and air pollution along with the aging of the population, and the intake of high-fat diets has become common due to the westernization of dietary life, and liver cancer and colon cancer due to a rapid increase in environmental pollutants and an increase in alcohol consumption. , Breast cancer, prostate cancer, etc. are on a continuous increase. In this situation, the creation of anticancer substances that can contribute to the promotion of human health, improvement of healthy quality of life, and improvement of human health by enabling early prevention and treatment of cancer is urgently required.

Among them, liver cancer is a cancer that ranks fourth among Korean men and sixth among women. It is a representative high-risk Korean predominant cancer, accounting for more than 22% of all cancer deaths in Korea (as of 2015). In addition, the treatment cost per patient is about 66.2 million won, which is the most common cancer among Koreans, and is a major disease that increases the socio-economic medical burden. Bayer's Nexavar is currently the only treatment for liver cancer, and it is forming a market of 1.2 trillion won per year through high growth of over 10% per year.

The diagnosis of liver cancer was mainly carried out as a surveillance test for a clear high-risk group, but less than 50% of patients diagnosed with liver cancer in Korea were detected by examination. To date, biomarkers using AFP and PIVKA II proteins have been developed for diagnosis of liver cancer, but development of high-performance biomarkers and diagnostic systems that can predict the treatment effect and prognosis for each liver cancer patient is in a state of sluggishness. In addition, in recent large-scale cancer genome projects such as TCGA (The Cancer Genome Atlas), large-scale genome/transcriptome/epigenetic analysis on cancer was performed to report multiple genome/transcriptome/epigenetic mutations and propose new molecular subtypes. However, biomarkers that are related to the prognosis of liver cancer patients have not 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 actively being conducted. Recently, as a type of new noncoding RNA, the existence of enhancer RNA (eRNA) produced by activated enhancer has been revealed. eRNAs are classified into 1D (unidirectional) eRNA and 2D (bidirectional) eRNA depending on the length and the way they are made. Unlike lncRNA, which has been actively studied recently, it is known that 5'capping occurs but no splicing or polyadenylation occurs.

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 present new targets for diagnosis and treatment of liver cancer. Moreover, efforts are needed to develop a liver cancer-specific prediction system by identifying 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 an integrated transcriptome analysis using liver cancer cell lines, and performed ChIP-sequencing for histone H3, H3K4me1 and H3K27ac to discover an enhancer activated in liver cancer cell lines, and ATAC- An integrated analysis was performed with enhancer peaks obtained from histone ChIP-seq by performing seq.

Accordingly, enhancers expressed from liver cancer cell lines were discovered, and AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261, STARD13-AS were used in The Cancer Genome Atlas (TCGA). As a result of analyzing the data, it was found that the number of liver cancer patients decreased compared to normal subjects.

Based on these experimental results, AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261, and STARD13-AS were used as specific biomarkers for liver cancer. It was confirmed that it could be 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 result.

One object of the present invention is any one or more biomarkers selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS. It is to provide a biomarker composition for diagnosis of liver cancer.

Another object of the present invention is any one or more eRNAs selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS (biomarker ) It is to provide a composition for diagnosis of liver cancer, including an agent for measuring the expression level of.

Including an agent measuring the expression level of any one or more eRNA selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS, It is to provide a kit for diagnosis of liver cancer.

Another object of the present invention is (a) from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS from isolated biological samples. Measuring the expression level of any one or more selected eRNAs;

(b) selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS of the measured expression level compared to Comparing to any one or more expression levels; And

(c) the expression level of any one or more selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS of the biological sample is compared If it is lower than the expression level of any one or more selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS of the normal control, it is determined as liver cancer. It is to provide a method of providing information for diagnosis of liver cancer, including the step.

In order to achieve the above object, the present invention provides any one or more biomarkers selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS. It provides a biomarker composition for diagnosis of liver cancer, including.

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

Liver cancer refers to a tumor in which hepatocytes lose their own function by continuous various stimulation and transform into cancer cells to achieve endless self-proliferation and spread to surrounding or distant places. Metastatic cancer has been transferred to. Primary liver cancer includes hepatocellular carcinoma caused by an abnormality in hepatocytes, cholangiocarcinoma caused by an abnormality in bile duct cells, and angiosarcoma, and more than 90% is hepatocellular carcinoma (HCC).

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

In general, the diagnosis and stage of cancer is determined by the type of cancer cells, the degree of differentiation, and the degree of metastasis through a tissue obtained by surgery or biopsy through a biopsy. In other words, the treatment and prognosis of general solid cancer are determined by the TNM stage.

However, in the case of liver cancer, it may be difficult to select a treatment method or predict the prognosis only with the TNM stage. This is because chronic liver disease occurs in most patients with liver cancer, and there is a limitation in selecting a treatment method even with advanced liver disease separate from cancer.

Therefore, when the marker according to the present invention is used, the onset of liver cancer can be monitored and diagnosed early. In the present invention, diagnosis means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is to confirm the onset of liver cancer, 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 of liver cancer, the disease state, the stage, the etiology, the presence or absence of complications, prognosis, and recurrence.

In the present invention, a biomarker is a substance that can be diagnosed by distinguishing liver cancer from normal hepatocytes, etc., and a polypeptide or nucleic acid showing an increase or decrease in liver cancer (e.g., eRNA, mRNA, etc.), lipid, glycolipid, glycoprotein, or sugar (monosaccharide , Disaccharides, oligosaccharides, etc.), and the like. For the purposes of the present invention, biomarkers for diagnosis of liver cancer are AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, which exhibit a specific low level of expression in liver cancer compared to normal liver cells. , LINC00261 and STARD13-AS any one or more eRNA selected from the group consisting of.

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

In the present invention, AC007319.1 (ENSG00000224063) can check the information by referring to https://www.genecards.org/cgi-bin/carddisp.pl?gene=ENSG00000224063.

In the present invention, AC073332.1 (ENSG00000237773) can be checked by referring to https://www.genecards.org/cgi-bin/carddisp.pl?gene=ENSG00000237773&keywords=ENSG00000237773.

AC103591.3 (ENSG00000273338) in the present invention can know the information by referring to https://www.genecards.org/cgi-bin/carddisp.pl?gene=ENSG00000273338&keywords=ENSG00000273338.

In the present invention, AL023755.1 (ENSG00000228697) can know the information by referring to https://www.genecards.org/cgi-bin/carddisp.pl?gene=ENSG00000228697&keywords=ENSG00000228697.

In the present invention, AL513327.1 (ENSG00000225313) can know the information by referring to https://www.genecards.org/cgi-bin/carddisp.pl?gene=ENSG00000225313&keywords=ENSG00000225313.

In the present invention, the information of LINC-PINT (long intergenic non-protein coding RNA, p53 induced transcript) (ENSG00000231721) is registered as GeneID: 378805 in the National Center for Biotechnology Information (NCBI).

In the present invention, the information of LINC00261 (long intergenic non-protein coding RNA 261) (ENSG00000259974) is registered as GeneID: 140828 in NCBI (National Center for Biotechnology Information).

In the present invention, the information of STARD13-AS (STARD13 antisense RNA) (ENSG00000236581) is registered as GeneID: 100874241 in NCBI (National Center for Biotechnology Information).

According to one embodiment of the present invention, AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261, STARD13-AS data from The Cancer Genome Atlas (TCGA) liver cancer sample As a result of analysis through, it was found that there was a decrease in liver cancer patients compared to normal subjects.

Based on these experimental results, AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261, and STARD13-AS were used as specific biomarkers for liver cancer. It was confirmed that it could be 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 measures the expression level of any one or more eRNAs selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS. It provides a composition for diagnosis of liver cancer comprising a formulation.

The expression level of one or more biomarkers selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS confirms the expression level of eRNA. You can know by doing it.

In the present invention, "measurement of eRNA expression level" is a process of checking the presence and expression level of the eRNA in a biological sample in order to diagnose liver cancer, and can be determined by measuring the amount of eRNA. Analysis methods for this are RT-PCR, Competitive RT-PCR (Competitive RT-PCR), Real-time RT-PCR (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting (Northern blotting) and DNA chips, but are not limited thereto.

The agent for measuring the eRNA level is preferably a primer pair or a probe, and the nucleic acid sequence of the gene of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261, STARD13-AS As this has been found, those skilled in the art can design primers or probes that specifically amplify specific regions of these genes based on the above sequences.

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 a base pair, and functions as a starting point for template strand copying. It means a short nucleic acid sequence. Primers can initiate synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature.

In the present invention, the sense and antisense primers of any one or more genes selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS are used. By performing PCR amplification, liver cancer can be diagnosed through whether or not a desired product is produced. The PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art.

In the present invention, the probe refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to several hundred bases capable of specific binding to an eRNA, and is labeled so that the presence or absence of a specific eRNA can be confirmed. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, using a probe complementary to any one or more polynucleotides selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS By performing hybridization, liver cancer can be diagnosed through hybridization. Selection of suitable probes and conditions for hybridization 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 can 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 homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkers (eg, methyl phosphonate, phosphotriester, Phosphoroamidate, carbamate, etc.) or to a charged linker (e.g., phosphorothioate, phosphorodithioate, etc.).

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

In addition, if necessary, since any known method that can check the amount of a marker can be used without limitation, ChIP (Chromatin Immunoprecipitation)-Seq, ChIP-qPCR, ATAC (Assay) can be used to analyze a large amount of multiple markers, that is, eRNA. 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 measures the expression level of any one or more eRNA selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS It provides a kit for diagnosis of liver cancer comprising a.

The kit of the present invention is any one or more selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS, which are biomarkers for diagnosis of liver cancer. It can be detected by checking the expression level of the biomarker, i.e. eRNA. The diagnostic kit of the present invention may include a primer and a probe for measuring the expression level of a liver cancer biomarker, as well as one or more other component compositions, solutions, or devices suitable for an analysis method.

Specifically, the kit for measuring the expression level of any one or more eRNA selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS It may be a kit containing the essential elements necessary to perform RT-PCR. In addition to each primer pair specific for the biomarker gene, the RT-PCR kit includes a test tube or other suitable container, reaction buffer (varies in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq- Enzymes such as polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like may be included. In addition, a primer pair specific to a gene used as a quantitative control may be included. Also preferably, the kit of the present invention may be a diagnostic kit containing essential elements necessary to perform a DNA chip. The DNA chip kit may include a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, agents, enzymes, etc. 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 amplifying a nucleic acid may be a polymerase, dNTP, a buffer, a nucleic acid, a coenzyme, a fluorescent substance, or a combination thereof. The polymerase is, for example, Taq polymerase. The kit may further include a reagent for separating nucleic acids. The reagent for separating the nucleic acid may include a cell lysis solution. The cell lysis solution may contain 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 a combination of two or more biomarkers alone, as in the above salpin composition, and a preferred example is the same as above. .

The present invention (a) the expression of any one or more selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS from the isolated biological sample Measuring the level;

(b) selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS of the measured expression level compared to Comparing to any one or more expression levels; And

(c) the expression level of any one or more selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS of the biological sample is compared If it is lower than the expression level of any one or more selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS of the normal control, it is determined as liver cancer. It provides a method of providing information for diagnosis of liver cancer, comprising the steps of.

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

In the present invention, the "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 expression level measurement, it is possible to diagnose the onset and progression of liver cancer in patients with suspected liver cancer by comparing the level of eRNA expression in the normal control group with the level of expression in the patients suspected of liver cancer.

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

Analysis methods for measuring eRNA levels include reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting, and DNA chip, but are not limited thereto. Through the above detection methods, it is possible to compare the expression level of eRNA in the normal control group with the level of eRNA expression in patients with suspected liver cancer, and determine whether a significant increase or decrease in the expression level of eRNA in the liver cancer biomarker gene is used to determine whether a patient suspected of liver cancer has occurred. 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 described above can be performed by electrophoresis after the reaction to check the band pattern and the thickness of the band, so that the expression and degree of eRNA of the gene used as a liver cancer biomarker can be checked. Can be diagnosed.

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

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

The present invention (a) the expression of any one or more selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS from the isolated biological sample Measuring the level;

(b) selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS of the measured expression level compared to Comparing to any one or more expression levels; And

(c) the expression level of any one or more selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS of the biological sample is compared If it is lower than the expression level of any one or more selected from the group consisting of AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS of the normal control, it is determined as liver cancer. step; And

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

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

The administration refers to introducing an anticancer agent to a subject by any suitable method, and the route of administration 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 AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS according to the present invention are excellent in the diagnosis of liver cancer. As it shows an effect, it can be usefully used for classification of liver cancer patients, selection of a treatment according to it, and establishment of a treatment plan.

1 shows a schematic diagram of the entire process for discovering liver cancer-specific enhancers and eRNAs.
2 shows the results of analyzing eRNA that is lower in liver cancer compared to the normal control group based on TCGA (The Cancer Genome Atlas) data.

Hereinafter, preferred examples and formulation examples are presented to aid in understanding the present invention. However, the following examples and formulation examples are provided for easier understanding of the present invention, and the contents of the present invention are 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 to discover liver cancer-specific enhancers and eRNAs.

HCC cell lines were fixed with 1% paraformaldehyde and glycine was added to stop the reaction. The pellet was dissolved in lysis buffer (5mM PIPES pH 8.0, 85nM 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 minutes until most pieces were in the range of 200 ~ 300bp. Antibodies were added for each immunoprecipitation and spun at room temperature for 2 hours to bind to beads (DynabeadsTM Protein A, Invitrogen). Antibodies used were H3 (2 μg per immune saliva, Abcam ab1791), H3K27ac (2 μg per immune saliva, Abcam ab4729) and H3K4me1 (2 μg per immune saliva, Abcam ab8895). For the control library, immunoprecipitation with 1 μg of non-specific IgG mouse antibody was used. Blocked antibody-conjugated beads were placed on a magnet, the supernatant was removed, the sonicated lysate was added to the beads, and incubated for 3-4 hours at 4 °C on a rotating machine. After washing the beads, the tags were reacted (Nextra® DNA Library Prep Kit, illumina). Wash the beads and cross-link formaldehyde by eluting them overnight at 65 °C with 1X TE buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA) containing 2.5 μl Proteinase K (Roche) and 1.5 μl 10% SDS. Was reverted. 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 the size was selected using AMPure XP beads to recover a library having a fragment length of 200-400 bp. RNA library sequencing was performed on the Illumina HiSeq2000 platform.

< Experimental example 2> ChIP - seq And Total RNA- seq analysis

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

Specifically, for the Chip seq, the lead quality was checked with'FastQC' and then lead filtering was performed with'Trimmomatic'. After performing alignment with'Bowtie2', a BAM file was created from which non-uniquely aligned reads and potential PCR duplicates were removed. After that, peack calling was performed using'MACS14', and peak annotation was performed with'HOMER'.

In addition, for RNA seq, read quality check was performed with'FastQC' and read filtering was performed with'Trimmomatic'. The total RNA-seq read was aligned to the Reference genom using'Tophat'. For the reference genome, Ensembl's GRCh38 was used. After that,'Cufflinks' was performed, and the expression level of the gene was calculated by FPKM.

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

H3K27 acetylation peaks, known to be high in the expressed enhancer, were selected on the entire genome in order to discover the enhancer specifically activated in the liver cancer cell line and the eRNA expressed therefrom. In order to discover only the activated enhancer, H3K27 acetylation peaks appearing in the gene promoter or inside were excluded. Next, 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, total RNA-seq data and integrated analysis were performed to discover eRNA expressed in the activated enhancer. A schematic diagram of a series of related analyzes is shown in FIG. 1.

< Experimental example 4> TCGA (AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC -PINT, LINC00261 And STARD13 -Any one selected from the group consisting of AS More than e Cancer Genome Atlas) data verification

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

<Experiment result>

One. In liver cancer cell lines Manifested eRNA Discovery and analysis

A total of 8 liver cancer cell lines were used to analyze the activated enhancer and eRNA expressed therefrom. Specifically, a total of 45 eRNAs were selected using 4 liver cancer cell lines (F-3-7, Huh-7-5, SNU182, PLC-PRF-5) having similar gene expression patterns among 8 liver cancer cell lines. The specific analysis results are shown in Table 1 below.

[Table 1]

As can be seen in Table 1, a total of 45 types of eRNA were found to be expressed in liver cancer cell lines. The eRNAs identified above included AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS.

2. TCGA Verification using data

The expression patterns of 45 eRNAs discovered using TCGA data were confirmed. A total of 8 eRNA expressions were decreased in liver cancer tissues (50 cases) compared to normal liver tissues (50 cases). Eight biomarker eRNAs discovered in liver cancer cell lines through this result are shown in FIGS. 2 and 2.

Decreased expression in liver cancer eRNA Decreased expression in liver cancer eRNA AC007319.1 AC073332.1 AC103591.3 AL023755.1 AL513327.1 LINC-PINT LINC00261 STARD13-AS

As can be seen in Table 2 and FIG. 2, it was confirmed that the expression of a total of 8 types of eRNA was reduced in liver cancer tissues compared to normal liver tissues.

Based on the above results, AC007319.1, AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS according to the present invention were significantly expressed in liver cancer tissues. Decreased, which showed a significant difference compared to the normal control group.

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

Claims (10)

A composition for diagnosis of liver cancer, comprising an agent measuring the level of eRNA expression of AC007319.1. The method of claim 1, further comprising an agent for measuring the expression level of any one or more eRNAs selected from the group consisting of AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS. To, a composition for diagnosis of liver cancer. 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 AC007319.1. The method of claim 1, wherein the eRNA expression level is measured by RT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA; RNase protection assay). ), Northern blotting (Northern blotting) and that 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 selected from the group consisting of ChIP-Seq, ChIP-qPCR, ATAC-Seq, DNase-seq, FAIRE-seq, CLIP-seq, RIP-seq, and luciferase assay. One or more, a composition for diagnosis of liver cancer. A kit for diagnosis of liver cancer, comprising the composition of any one of claims 1 to 5. (a) measuring the expression level of AC007319.1 from the isolated biological sample;
(b) comparing the measured expression level with the expression level of AC007319.1 of the compared normal control sample; And
(c) determining as liver cancer when the expression level of AC007319.1 in the biological sample is lower than the expression level of AC007319.1 in the contrasting normal control; comprising, providing information for diagnosis of liver cancer. The expression level of claim 7, wherein (a) any one or more expression levels selected from the group consisting of AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS from the isolated biological sample. Measuring a;
(b) any one or more expressions selected from the group consisting of AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS in the normal control sample compared to the measured expression level Comparing with the level; And
(c) AC007319 of a normal control in which the expression level of any one or more selected from the group consisting of AC073332.1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and STARD13-AS of the biological sample is compared .1, AC103591.3, AL023755.1, AL513327.1, LINC-PINT, LINC00261 and if it is lower than the expression level of any one or more selected from the group consisting of STARD13-AS, determining as liver cancer; liver cancer further comprising How to provide information for diagnosis. The method of claim 7, wherein the measurement of the expression level in step (a) is to measure the expression level of eRNA. The method of claim 7, wherein the measurement of the expression level in step (a) is RT-PCR, competitive 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 method for providing information for liver cancer diagnosis.

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