KR102238258B1 - A novel biomarker for diagnosing liver cancer and a treating method using thereof - Google Patents

Abstract

The present invention provides a liver cancer diagnostic biomarker composition comprising LINC01572, and a liver cancer therapeutic agent comprising an inhibitor thereof.
The biomarkers according to the present invention have excellent effects in diagnosing liver cancer, and thus can be usefully used for classification of liver cancer patients, selection of therapeutic agents according to them, and establishment of treatment plans. In addition, when it is suppressed by targeting it, it exhibits an excellent effect on liver cancer.

Description

A novel biomarker for liver cancer diagnosis and a treatment method using the same {A NOVEL BIOMARKER FOR DIAGNOSING LIVER CANCER AND A TREATING METHOD USING THEREOF}

The present invention relates to a novel biomarker composition for diagnosing liver cancer, a method for diagnosing liver cancer using the same, and to a use of liver cancer treatment comprising an inhibitor for the biomarker.

Cancer has a high mortality rate worldwide, and is the most common cause of death in Western society after cardiovascular disease. 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 diet is generalized due to the westernization of diet, and liver cancer and colon cancer due to the rapid increase of environmental pollutants and the 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 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 on average.

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, recent large-scale cancer genome projects such as TCGA (The Cancer Genome Atlas) performed large-scale genome/transcriptome/epigenetic analysis on cancer to report multiple genome/transcript/epigenetic mutations and suggest 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.

Accordingly, 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.

On the other hand, the liver plays a role of filtering most of the blood in the body and alleviating the action of drugs that can exhibit various toxicities. When cancer occurs in the liver, the types of anticancer drugs that can be used are very limited due to toxicity problems. Accordingly, in general, Nexavar (Sorafenib) is used as a therapeutic agent, but the therapeutic effect is not sufficiently satisfactory compared to other anticancer drugs, and thus there are quite a lot of demands for new drugs. In particular, in the case of liver cancer, when it reaches stage 3 or stage 4, distant metastasis occurs a lot, and cancer metastasis to the lungs or other organs occurs a lot. For this metastasis, use of drugs due to the above-mentioned problems in the liver itself. Because this is very limited, treatment for metastasis is not resolved, resulting in the death of a large number of patients.

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- By performing seq, an integrated analysis was performed with enhancer peaks obtained from histone ChIP-seq.

Accordingly, as a result of discovering an enhancer activated from a liver cancer cell line, and analyzing LINC01572 through the data of The Cancer Genome Atlas (TCGA), it was found that it was increased in liver cancer patients compared to normal subjects.

When reviewing based on these experimental results, it was confirmed that LINC01572 can be used as a specific biomarker for liver cancer, and the present invention was completed.

In particular, it was confirmed that the biomarkers according to the present invention showed the same results in ChIP-seq analysis and TCGA, and thus excellent effects as markers are recognized.

In addition, the biomarker according to the present invention can provide more accurate diagnostic information in that it can confirm the same expression pattern not only in LINC01572 itself, but also in the TAD group and the target gene based on expression correlation, and further include related groups.

In more detail, the expression correlation-based target gene is known to play an important role in regulating gene expression, particularly in activating the expression of the target gene, so the expression level of eRNA and the expression level of the target gene are very high. Possibility to show correlation. Therefore, genes showing the same expression change pattern as each eRNA (increase or decrease in expression) can be selected and reviewed as target genes based on expression correlation. Genes showing the same expression pattern detected here were selected as target genes based on expression correlation. In other words, since eRNA can act as a regulator, mRNA having a significant correlation with the expression level of eRNA was selected through a correlation test and defined as a target gene for eRNA.

In addition, it was confirmed that when LINC01572 is specifically inhibited, it exhibits excellent therapeutic efficacy in liver cancer.

One object of the present invention is to provide a biomarker composition for diagnosis of liver cancer comprising the LINC01572 biomarker.

Another object of the present invention is to provide a composition for diagnosis of liver cancer, including an agent measuring the expression level of LINC01572 eRNA (biomarker).

It is to provide a kit for diagnosis of liver cancer, including an agent measuring the expression level of the eRNA of LINC01572.

Another object of the present invention is (a) measuring the eRNA expression level of LINC01572 from the isolated biological sample;

(b) comparing the measured expression level with the expression level of LINC01572 in the compared normal control sample; And

(c) If the expression level of LINC01572 in the biological sample is higher than the expression level of LINC01572 in the contrasting normal control, it is to provide a method of providing information for diagnosis of liver cancer, including determining as liver cancer.

It provides a pharmaceutical composition for the prevention or treatment of liver cancer, comprising the LINC01572 inhibitor.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosis of liver cancer comprising the biomarker of LINC01572.

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 intrinsic function by continuous various stimulation and transform into cancer cells to achieve endless self-proliferation and spread to surrounding or distant places.It occurs in the primary tumor and in the liver, resulting in other organs. Metastatic cancer has been transferred to. Primary liver cancer includes hepatocellular carcinoma caused by an abnormality in hepatocyte cells, 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. That is, 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 the selection of treatment methods only 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 determine whether 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 name of the disease, and may include the name of the liver cancer, the state of the disease, 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.), lipids, glycolipids, glycoproteins or sugars (monosaccharides , Disaccharides, oligosaccharides, etc.), and the like. For the purposes of the present invention, the biomarker for diagnosis of liver cancer is an eRNA of LINC01572 that exhibits a specifically high level of expression in liver cancer compared to normal liver cells.

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, LINC01572 (long intergenic non-protein coding RNA 1572) (ENSG00000261008) information is registered as GeneID: 101927957 in NCBI (National Center for Biotechnology Information).

According to one embodiment of the present invention, as a result of analyzing LINC01572 through the data liver cancer sample of The Cancer Genome Atlas (TCGA), it was found that the increase in liver cancer patients compared to the normal person.

When reviewing based on these experimental results, it was confirmed that LINC01572 can be used as a specific biomarker for liver cancer, and the present invention was completed.

In the present invention, the biomarker composition may be used alone, or may be used in combination with other liver cancer markers.

For example, the biomarker composition according to the present invention may further include PMFBP1, ZNF821, or both as a marker as a gene in Topologically Associated domains (TAD) to which LINC01572 eRNA belongs. That is, as mRNA in the TAD identical to LINC01572 according to the present invention, PMFBP1 (polyamine modulated factor 1 binding protein 1, GeneID: 83449) and ZNF821 (zinc finger protein 821, GeneID: 55565) are similar to LINC01572, compared to normal samples. It is highly expressed in samples.

In addition, as mRNA related to LINC01572 eRNA, C14orf93 (chromosome 14 open reading frame 93, GeneID: 60686), CEP152 (centrosomal protein 152, GeneID: 22995), DXO (decapping exoribonuclease, GeneID: 1797), HELLS (helicase, lymphoid specific, GeneID: 3070), NCAPH2 (non-SMC condensin II complex subunit H2, GeneID: 29781), NFKB2 (nuclear factor kappa B subunit 2, GeneID: 4791), NMB (neuromedin B, GeneID: 4828), SFSWAP (splicing factor SWAP) , GeneID: 6433), STK19 (serine/threonine kinase 19, GeneID: 8859) and TIGD5 (tigger transposable element derived 5, GeneID: 84948) may further include any one or more selected from the group consisting of a marker. In other words, with respect to the LINC01572 eRNA, C14orf93, CEP152, DXO, HELLS, NCAPH2, NFKB2, NMB, SFSWAP, STK19, TIGD5 are mRNAs with high correlation with TCGA LIHC Tumor. Similar to LINC01572, they are higher in liver cancer samples compared to normal samples. Is expressed.

Since the mRNA in the TAD and related mRNA (target genes based on expression correlation) show the same similar expression pattern in the LINC01572 eRNA and liver cancer and normal tissues, more accurate diagnostic information can be provided. That is, the aforementioned mRNAs are highly expressed in liver cancer when compared to normal liver tissue.

The present invention also provides a composition for diagnosis of liver cancer, comprising an agent measuring the expression level of eRNA of LINC01572.

The expression level of the biomarker of LINC01572 can be determined by checking the expression level of eRNA.

In the present invention, "measurement of eRNA and/or mRNA expression level" refers to a process of checking the presence and expression level of the eRNA and/or mRNA in a biological sample to diagnose liver cancer. I can. 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. blotting) and DNA chips, but are not limited thereto.

The agent for measuring the level of eRNA and/or mRNA is preferably a primer pair or a probe, and since the nucleic acid sequence of the gene of LINC01572 has been identified, those skilled in the art have a primer or probe that specifically amplifies a specific region of these genes based on the sequence. Can design.

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, for example, by performing PCR amplification using the sense and antisense primers of the gene of LINC01572, 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, a probe refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to a few hundred bases that can achieve specific binding with eRNA and/or mRNA, and is labeled You can check the presence or absence. 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. For example, in the present invention, hybridization is performed using a probe complementary to the polynucleotide of LINC01572, and 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 (eg phosphorothioate, phosphorodithioate, etc.).

Examples of formulations and methods for measuring the level of LINC01572 may be equally applied to PMFBP1 ZNF821, C14orf93, CEP152, DXO, HELLS, NCAPH2, NFKB2, NMB, SFSWAP, STK19 and TIGD5.

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

For example, the composition for diagnosis of liver cancer according to the present invention may further include an agent measuring the expression level of the mRNA of PMFBP1, ZNF821, or both of them or the protein thereof as a gene in the TAD to which LINC01572 eRNA belongs.

In addition, LINC01572 eRNA-related mRNA further includes an agent for measuring the expression level of any one or more mRNAs or proteins thereof selected from the group consisting of C14orf93, CEP152, DXO, HELLS, NCAPH2, NFKB2, NMB, SFSWAP, STK19 and TIGD5. can do.

Since the gene and related mRNA in the TAD exhibit the same similar expression pattern as LINC01572 eRNA in liver cancer and normal tissues, more accurate diagnostic information can be provided.

In addition, if necessary, since any known method that can check the amount of the marker can be used without limitation, ChIP-Seq, ChIP-qPCR, ATAC-Seq, DNase-seq for the analysis of a large amount of multiple markers, that is, eRNA. , FAIRE-seq, CLIP-seq, RIP-seq, and any one or more selected from the group consisting of luciferase assay may be used, but is not limited thereto.

In addition, the measurement of the expression level in consideration of the mRNA may be performed using an agent for measuring the protein expression level thereof. That is, for PMFBP1 ZNF821, C14orf93, CEP152, DXO, HELLS, NCAPH2, NFKB2, NMB, SFSWAP, STK19 and TIGD5, it is also possible to measure its expression change as an agent measuring its protein expression level.

"Measurement of protein level" is a process of confirming the presence and level of expression of a protein expressed from a marker gene in a biological sample to diagnose cancer. Specifically, an antibody that specifically binds to the protein of the gene is Can be used to determine the amount of protein.

In the present invention, "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes all of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Since the marker protein has been identified as described above, generating an antibody using it can be easily prepared using techniques well known in the art.

The polyclonal antibody can be produced by a method well known in the art in which the above-described marker protein antigen is injected into an animal and blood is collected from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, cow and dog.

Monoclonal antibodies are a hybridoma method well known in the art (see Kohler and Milstein (1976) European Jounral of Immunology 6:511-519), or a phage antibody library (Clackson et al, Nature, 352: 624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). The antibody prepared by the above method can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.

In addition, the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules. A functional fragment of an antibody molecule refers to a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.

As an analysis method to determine the amount of the target protein bound to it using the antibody, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, OY Ouchterlony immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), protein There is a chip (protein chip) and the like, but is not limited thereto.

The present invention also provides a kit for diagnosis of liver cancer, comprising an agent for measuring the expression level of eRNA of LINC01572.

The kit of the present invention can be detected by checking the biomarker of LINC01572, a biomarker for diagnosis of liver cancer, that is, the expression level of 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 constituent compositions, solutions, or devices suitable for an analysis method.

Specifically, the kit for measuring the eRNA expression level of LINC01572 may be a kit including essential elements necessary for performing 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 inhibitor, DEPC-water, sterile water, and the like. 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, and the like for preparing a fluorescently labeled probe. In addition, the substrate may include a 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 comprises: eRNA for a combination of two or more biomarkers alone, as in the above salpin composition; And an agent for measuring the expression level of the mRNA or protein thereof in the related mRNA or TAD, and a preferred example is as described above.

As for the measurement of the expression level of mRNA, the previous information on the measurement of the expression level of eRNA can be appropriately applied.

Accordingly, as a gene in the TAD to which the LINC01572 eRNA belongs, it may include an agent for measuring the expression level of the mRNA of PMFBP1, ZNF821 or both, or a protein thereof, and/or the mRNA associated with the LINC01572 eRNA, C14orf93, CEP152, DXO, HELLS, NCAPH2, NFKB2, NMB, SFSWAP, STK19 and TIGD5 may comprise a kit further comprising an agent for measuring the expression level of any one or more mRNA or protein thereof selected from the group consisting of.

The kits measured and provided based on eRNA and mRNA are the same as the salpin bars above.

Meanwhile, when considering the protein of the mRNA marker as a basis, a kit using an agent for measuring the protein expression level thereof may be further included and provided. For example, it may contain the necessary elements necessary to perform an ELISA. The ELISA kit contains antibodies specific for the marker protein. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity with other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. In addition, the ELISA kit may contain an antibody specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or antibodies capable of binding. Other materials may be included.

The present invention comprises the steps of: (a) measuring the expression level of LINC01572 from the isolated biological sample;

(b) comparing the measured expression level with the expression level of LINC01572 in the compared normal control sample; And

(c) If the expression level of LINC01572 in the biological sample is higher than the expression level of LINC01572 in the contrasting normal control, it provides a method of providing information for diagnosis of liver cancer, including determining as liver cancer.

The expression levels of steps (a) and (b) can be detected at the eRNA level, and eRNA separation 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 measurement of the expression level, 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 it is expressed more 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, but are not limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting, and DNA chip. Through the above detection methods, it is possible to compare the eRNA expression level in the normal control and the eRNA expression level in the suspected liver cancer patient, and determine whether a significant increase in the expression level of the eRNA in the liver cancer biomarker gene is determined to determine whether the onset of a suspected liver cancer patient. 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 to check the expression and degree of eRNA expression of the 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 its fragment is attached at high density to a glass-like substrate, and the eRNA is separated from the sample, and the end or the inside thereof is a cDNA probe labeled with a fluorescent substance. 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 the information for diagnosis of liver cancer may include, without limitation, measurement of the expression level for a combination of two or more biomarkers as in the salpin composition, and a preferred example is as described above.

In addition to the measurement of the eRNA of step (a) mentioned above, any one or more mRNAs selected from the group consisting of PMFBP1 ZNF821, C14orf93, CEP152, DXO, HELLS, NCAPH2, NFKB2, NMB, SFSWAP, STK19 and TIGD5, or their It may include the step of measuring the expression level of the protein. Accordingly, (b) any one or more mRNAs selected from the group consisting of PMFBP1 ZNF821, C14orf93, CEP152, DXO, HELLS, NCAPH2, NFKB2, NMB, SFSWAP, STK19 and TIGD5 of the normal control sample compared to the measured expression level Or comparing the level of expression of the protein thereof; And (c) PMFBP1 ZNF821, C14orf93, CEP152, DXO, HELLS, NCAPH2, NFKB2, NMB, SFSWAP, STK19 and TIGD5 of the biological sample. If it is higher than the expression level of LINC01572 of the control group, it may include the step of determining as liver cancer.

The above measurement and determination method can be applied by appropriately modifying the above salpin content.

The present invention comprises the steps of: (a) measuring the expression level of LINC01572 from the isolated biological sample;

(b) comparing the measured expression level with the expression level of LINC01572 in the compared normal control sample; And

(c) determining as liver cancer when the expression level of LINC01572 in the biological sample is higher than the expression level of LINC01572 in the compared normal control group; 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, drug activity, 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 of 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, such as oral or parenteral, as long as it can reach the target tissue.

In addition, as mentioned above, it may further include confirmation and determination of the expression level of the related mRNA or mRNA in the TAD in each step.

The present invention also provides a pharmaceutical composition for preventing or treating liver cancer comprising an inhibitor of LINC01572 expression.

By inhibiting LINC01572 expression, it can exhibit excellent effects such as increased hepatocyte death, change in metastasis of cells, that is, inhibition of change in migration and invasion ability.

In the present invention, the term "LINC01572 inhibitor" is used as a collective term for all agents that reduce the expression or activity of LINC01572, specifically, it affects the expression of LINC01572 decreases, acts directly on LINC01572, or acts indirectly on its ligand. Any agent that reduces the activity of LINC01572 by reducing the expression level of LINC01572 at the level of transcription, mRNA, or translation, or by interfering with LINC01572 activity, may be included.

The LINC01572 inhibitor may be used without limitation in its form, such as a compound capable of inhibiting the expression of LINC01572 or activity by targeting LINC01572, a nucleic acid, a peptide, a virus, or a vector containing the nucleic acid. The LINC01572 inhibitor is, but is not limited to, siRNA or shRNA that degrades the mRNA of the LINC01572 gene, and antisense oligonucleotides that reduce the expression of the LINC01572 protein. In addition, as a LINC01572 inhibitor that binds to the LINC01572 protein and inhibits its function, it may be an aptamer or a low molecular weight compound.

In an exemplary embodiment, the antisense oligonucleotide may have SEQ ID NO: 1.

In the present invention, the term "treatment" refers to clinical intervention in order to alter the natural process of an individual or cell to be treated, and this may be performed during or to prevent a clinical pathological condition. The desired therapeutic effect includes preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, and alleviating the disease state. Or temporary alleviation, remission or improving prognosis. Preferably, the present invention includes all actions of improving the course of liver cancer by administering a composition containing a substance that inhibits LINC01572. In addition, "prevention" refers to any action of inhibiting or delaying the onset of liver cancer by administration of a composition containing a substance that inhibits LINC01572 according to the present invention.

In the present invention, the term "antisense oligonucleotide" refers to DNA, RNA, or derivatives thereof containing a nucleic acid sequence that is complementary to a sequence of a specific RNA, and inhibits the action of RNA by binding to a complementary sequence in the RNA. The antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to the LINC01572 eRNA and capable of binding to the eRNA. This can inhibit the translation, translocation, maturation, or all other essential activity of the LINC01572 eRNA into the entire biological function. The length of the antisense oligonucleotide may be 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases. The antisense oligonucleotide may be synthesized in vitro by a conventional method and administered in vivo, or the antisense oligonucleotide may be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is the use of RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow antisense RNA to be transcribed using a vector in which the origin of the multicloning site (MCS) is in the opposite direction. It is preferable that the antisense RNA has a translation stop codon in the sequence so that it is not translated into a peptide sequence. The design of the antisense oligonucleotide that can be used in the present invention can be prepared according to a method known in the art with reference to the nucleotide sequence of LINC01572.

In the present invention, the term "aptamer" is a single-stranded oligonucleotide, has a size of about 20 to 60 nucleotides, and refers to a nucleic acid molecule having binding activity to a predetermined target molecule. It has a variety of three-dimensional structures depending on the sequence, and can have high affinity with a specific substance, such as an antigen-antibody reaction. The aptamer can inhibit the activity of a predetermined target molecule by binding to a predetermined target molecule. The aptamer of the present invention may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may also be in a linear or cyclic form. Preferably, the aptamer may play a role of inhibiting the activity of LINC01572 by binding to LINC01572. Such an aptamer can be prepared from the sequence of LINC01572 by a method known to those skilled in the art.

In the present invention, the terms "siRNA" and "shRNA" are nucleic acid molecules capable of mediating RNA interference or gene silencing, and because they can inhibit the expression of a target gene, an efficient gene knock down method or Used as a gene therapy method. The shRNA is a hairpin structure formed by binding between complementary sequences in a single-stranded oligonucleotide, and in vivo, the shRNA is a small RNA fragment having a size of 21 to 25 nucleotides while being cleaved by a dicer. It becomes siRNA, which is a double-stranded oligonucleotide, and can specifically bind to mRNA having a complementary sequence to inhibit its expression. Therefore, which means of shRNA and siRNA to be used can be determined by the choice of those skilled in the art, and if the mRNA sequences targeted by them are the same, a similar expression reducing effect can be expected. For the purpose of the present invention, the LINC01572 can be inhibited by specifically acting on LINC01572 and cleaving the LINC01572 mRNA molecule to induce RNA interference (RNAi). siRNA can be synthesized chemically or enzymatically. The siRNA production method is not particularly limited, and a method known in the art may be used. For example, a method for synthesis of siRNA directly chemically, in vitro (in vitro) Synthesis of siRNA using the transcription, in vitro method of cutting using a long double-stranded RNA of the enzyme synthesized by the (in vitro) transcription, Expression methods through intracellular delivery of shRNA expression plasmids or viral vectors, and expression methods through intracellular delivery of a polymerase chain reaction (PCR) induction siRNA expression cassette are not limited thereto.

The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions. Compositions containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration may include tablet pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, or lactose in one or more compounds. It can be prepared by mixing (lactose), gelatin, and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

In addition, the pharmaceutical composition of the present invention is not limited thereto, but tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations And it may have any one formulation selected from the group consisting of suppositories.

Another aspect of the present invention for achieving the above object is to provide a method for treating liver cancer comprising administering to an individual a pharmaceutical composition comprising a LINC01572 inhibitor as an active ingredient.

In the present invention, the term "individual" refers to all animals, including humans, possessing or developing liver cancer disease of the present invention, and may be an individual other than humans. By administering the pharmaceutical composition of the present invention to an individual, it exhibits excellent effects in the treatment of liver cancer and can also inhibit metastasis of liver cancer.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

In the present invention, the term "administration" refers to introducing the pharmaceutical composition of the present invention to a subject by any suitable method, and the route of administration may be administered through various routes, either orally or parenterally, as long as it can reach the target tissue.

The pharmaceutical composition may be appropriately administered to a subject according to a conventional method, route of administration, and dosage used in the art, depending on the purpose or need. Examples of the route of administration may be oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, an appropriate dosage and number of administrations may be selected according to methods known in the art, and the amount and number of administrations of the pharmaceutical composition of the present invention to be actually administered are the type of symptom to be treated, the route of administration, sex, health status, It can be appropriately determined by various factors such as diet, age and weight of the individual, and the severity of the disease.

The term "pharmaceutically effective amount" in the present invention means an amount sufficient to suppress or alleviate the disease in a reasonable ratio applicable to medical use, and the effective dose level is the type and severity of the subject, age, sex, and activity of the drug. , Sensitivity to drugs, 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 of the above factors, and can be easily determined by a person skilled in the art. For example, the pharmaceutically effective amount is 0.5 to 1000 mg/day/kg of body weight, preferably 0.5 to 500 mg/day/kg of body weight.

Another aspect of the present invention for achieving the above object is to provide a use of a composition comprising a LINC01572 inhibitor in the manufacture of a medicament for the treatment of liver cancer.

Another aspect of the present invention for achieving the above object is to provide a composition comprising a LINC01572 inhibitor for use in the treatment of liver cancer.

The biomarker of LINC01572 according to the present invention and genes related thereto show excellent effects in diagnosis of liver cancer, and thus can be usefully used for classification of liver cancer patients, selection of therapeutic agents according to this, and establishment of treatment plans.

1 shows a schematic diagram of the entire process for discovering a liver cancer-specific enhancer and eRNA.
2 shows the results of analyzing eRNA that is highly expressed in liver cancer compared to the normal control group based on TCGA (The Cancer Genome Atlas) data.
3 shows a box plot result confirming the difference in the expression level and significance of the gene in TAD of LINC01572, which is highly expressed in liver cancer compared to the normal control group, based on TCGA (The Cancer Genome Atlas) data.
FIG. 4 is a diagram showing differences in the expression levels, significance, and expression patterns of genes in TAD of LINC01572, which is highly expressed in liver cancer compared to the normal control group, based on TCGA (The Cancer Genome Atlas) data.
FIG. 5 shows a box plot result confirming the difference in the expression level and significance of the lower target gene confirmed through an expression correlation analysis with LINC01572, which is highly expressed in liver cancer compared to the normal control group, based on TCGA (The Cancer Genome Atlas) data.
6 is a diagram showing the difference in expression levels, significance, and expression patterns of sub-target genes identified through expression correlation analysis with LINC01572, which is highly expressed in liver cancer compared to the normal control group based on TCGA (The Cancer Genome Atlas) data.
7 shows the results of confirming the inhibitory efficacy of antisense oligo treatment for LINC01572.
8 shows the results of confirming that LINC01572 inhibits cell growth according to Knock down.

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 of the fragment size was 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, and the sonicated lysate was added to the beads, followed by incubation at 4 °C for 3 to 4 hours 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 at 65 °C overnight 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

To discover activated enhancers, ChIP-seq for H3K4 monomethylation (H3K4me1) and H3K27 acetylation was performed. 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. Afterwards, peack calling was performed using'MACS14', and peak annotation was performed with'HOMER'.

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

<Experimental Example 3> Discovery of liver cancer-specific enhancer and eRNA

H3K27 acetylation peaks, known to be high in the activated enhancer, were selected on the entire genome in order to discover the enhancer specifically activated in liver cancer cell lines and the eRNA expressed therefrom. In order to discover only the activated enhancer, H3K27 acetylation peaks appearing in the gene's 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> Selection of lower target genes of eRNA

1) Selection of lower target genes through TAD analysis

Topologically associating domain (TAD) means that the enhancer that regulates gene expression and the target gene whose expression is regulated are contained in the same space even within the chromosome. TAD analysis was performed to select the target gene of the selected eRNA, and through this, target genes expected to be regulated by each eRNA were selected.

2) Correlation-based identification of target genes

eRNA is known to play an important role in regulating gene expression, particularly in activating the expression of a target gene. Therefore, it is possible to show a very high correlation between the expression level of eRNA and the expression level of the target gene. Therefore, genes showing the same expression change pattern as each eRNA (increased or decreased expression) were selected and selected as target gene candidates.

<Experimental Example 5> Verification using TCGA (The Cancer Genome Atlas) data

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.

< Experimental example 6> To LINC01572 About antisense oligo Efficacy verification related

The antisense oligonucleotide (ASO) for LINC01572 was custom-designed and purchased from Qiagen. The ASO is complementary to the nucleotide sequence (5'-CAGTCTGTCTCTATGT-3'- SEQ ID NO: 2 ) located in the 6th exon of the LINC01572 gene, and its sequence is as follows (5'-ACATAGAGACAGACTG-3'- sequence Number 1 ).

To transfect this customized ASO, lipofectamine RNAimax (invitrogen) was used. At the time of transfection, customized ASO (20nM) and lipofectamine RNAimax (based on 2ul-24well) were mixed and treated on FT 3-7 cells. The first transfection was performed and the second transfection was performed 24 hours later, and the cells were harvested after 48 hours (a total of 72 hours incubation). The total RNA of the cell was isolated using the RNeasy Mini Kit (qiagen), and the knock-down efficiency of the gene was confirmed using real time PCR.

< Experimental example 7>LINC01572 Confirmation of cell growth inhibition by knock down

Through two transfections, the FT3-7 cells in which LINC01572 was effectively knocked down were seeded again in 24 wells, and the cell growth was confirmed from the day of seeding to 72 hours.

<Experimental Results>

1. Identification of eRNA expressed in liver cancer cell lines and analysis using 4 liver cancer cell lines

A total of 6 eRNAs were discovered by analyzing the activated enhancer and the eRNA expressed therefrom using a total of 8 liver cancer cell lines.

Then, 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. It was confirmed that 6 of the eRNAs were expressed in all 8 liver cancer cell lines.

The specific analysis results are shown in Table 1 below.

[Table 1]

As can be seen in Table 1, it was confirmed that all six eRNAs of THUMPD3-AS1, TRAF3IP2-AS1, LINC00511, SAP30L-AS1, LINC01572, and AL136311.1 were expressed in all eight hepatic cancer cell lines.

2. Verification using TCGA data

The expression patterns of 45 eRNAs discovered using TCGA data were confirmed. A total of 11 eRNAs were expressed in liver cancer tissues (50 cases) compared to normal liver tissues (50 cases). As can be seen in FIG. 2, among them, LINC01572, in particular, exhibited excellent diagnostic effect (ie, an expression difference having a very significant meaning between liver cancer and a normal control group) was exhibited.

3. Identification of eRNA sub-target genes

1) Selection of lower target genes through TAD analysis

The results of analyzing the gene expression pattern in the TAD to which the eRNA belongs are shown in Table 2 below and FIGS. 3 and 4. Genes with Q <0.01 and normal.mean <tumor.mean were selected. The significance value indicated in the box plot is the Q value (adjusted P).

eRNA mRNA TCGA
normal.mean TCGA
tumor.mean adjust.p LINC01572 PMFBP1 3.42E-02 1.21E-01 5.40E-03 LINC01572 ZNF821 3.53E-01 4.73E-01 6.78E-04

As can be seen in FIGS. 3 and 4, it was confirmed that the same expression pattern (ie, increased in liver cancer compared to the normal control group) was shown for PMFBP1 and ZNF821, and it was highly likely to be a direct sub-target gene of LINC01572.

2) Subordinate through expression correlation analysis Target gene Selection

eRNA same expression pattern for Reviewing the (increased or decreased expression) and by screening a gene showing the same expression change pattern selected for this, a target gene candidate in 10 species of a total of 16 genes (that is, in the control group compared to liver Increase, 62.5%).

The results are shown in Table 3 and FIGS. 5 and 6. Genes with Q <0.01 and normal.mean <tumor.mean were selected. The significance value indicated in the box plot is the Q value (adjusted P).

eRNA mRNA TCGA
normal.mean TCGA
tumor.mean adjust.p Pval Cor R.squared LINC01572 C14orf93 9.18E-01 1.30E+00 1.00E-09 2.78E-03 9.05E-01 7.29E-01 LINC01572 CEP152 1.36E-01 2.76E-01 3.07E-05 3.97E-04 -1.00E+00 9.80E-01 LINC01572 DXO 2.02E+00 2.48E+00 5.30E-07 2.78E-03 -9.05E-01 7.82E-01 LINC01572 HELLS 1.51E-01 4.91E-01 6.80E-08 2.78E-03 9.05E-01 8.36E-01 LINC01572 NCAPH2 2.75E+00 3.33E+00 3.27E-09 2.78E-03 9.05E-01 7.98E-01 LINC01572 NFKB2 2.92E+00 3.55E+00 3.92E-05 2.78E-03 -9.05E-01 7.69E-01 LINC01572 NMB 8.69E-01 1.71E+00 1.34E-06 2.78E-03 9.05E-01 8.24E-01 LINC01572 SFSWAP 1.21E+00 1.52E+00 1.69E-08 2.78E-03 -9.05E-01 7.95E-01 LINC01572 STK19 1.13E+00 1.40E+00 1.94E-05 2.78E-03 -9.05E-01 8.31E-01 LINC01572 TIGD5 7.46E-01 1.49E+00 1.70E-11 2.78E-03 -9.05E-01 9.73E-01

As can be seen in FIGS. 5 and 6, 10 genes showing the same expression pattern in the expression correlation analysis were identified as C14orf93, CEP152, DXO, HELLS, NCAPH2, NFKB2, NMB, SFSWAP, STK19, and TIGD5.

Through the above results, it was confirmed that not only LINC01572 but also a lower target gene through an expression correlation analysis with a gene in its TAD can be used as a diagnostic marker for liver cancer.

4. To LINC01572 About antisense oligo Confirmation of inhibitory efficacy through treatment

Fig. 7 shows the results of confirming the inhibitory efficacy through antisense oligo (SEQ ID NO: 1) treatment for LINC01572. As can be seen in Figure 7, the expression of LINC01572 was greatly reduced, and its expression was clearly confirmed to be suppressed.

5. Confirmation that LINC01572 inhibits cell growth by knock down

After re-seeding the FT3-7 cells in which LINC01572 was knocked down in 24 well, growth of the cells was confirmed from the day of seeding until 72 hours. The results are shown in FIG. 8.

As can be seen in Figure 8, it was confirmed that the growth of the cells was significantly decreased by 25% compared to normal cells 3 days after observation.

Through this, it was confirmed that the inhibition of LINC01572 has an excellent effect on inhibiting the growth of cancer cells, especially liver cancer.

<110> NATIONAL CANCER CENTER EWHA UNIVERSITY-INDUSTRY COLLABORATION FOUNDATION INDUSTRY-UNIVERSITY COOPERATION FOUNDATION HANYANG UNIVERSITY ERICA CAMPUS KNU-INDUSTRY COOPERATION FOUNDATION INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY <120> A NOVEL BIOMARKER FOR DIAGNOSING LIVER CANCER AND A TREATING METHOD USING THEREOF <130> ewha189p <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> 1 <400> 1 acatagagac agactg 16 <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> 2 <400> 2 cagtctgtct ctatgt 16

Claims (12)

A composition for diagnosis of liver cancer, comprising an agent measuring the level of eRNA expression of LINC01572. 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 eRNA. 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. According to claim 1, PMFBP1 ZNF821, C14orf93, CEP152, DXO, HELLS, NCAPH2, NFKB2, NMB, SFSWAP, STK19 and TIGD5 any one or more selected from the group consisting of an agent for measuring the expression level of mRNA or protein thereof is further prepared. Containing, 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 LINC01572 from the isolated biological sample;
(b) comparing the measured expression level with the expression level of LINC01572 in a contrast normal control sample; And
(c) If the expression level of LINC01572 in the biological sample is higher than the expression level of LINC01572 in the contrasting normal control, the method of providing information for diagnosis of liver cancer comprising the step of determining as liver cancer. 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 a method for providing information for diagnosis of liver cancer by any one selected from the group consisting of a DNA chip. A pharmaceutical composition for the prevention or treatment of liver cancer comprising a LINC01572 expression inhibitor selected from the group consisting of siRNA, shRNA and antisense oligonucleotides. delete The pharmaceutical composition for preventing or treating liver cancer according to claim 10, wherein the antisense oligonucleotide has the nucleotide sequence of SEQ ID NO: 1.

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