KR101704533B1 - Err as the biomaker to liver cancer and use thereof - Google Patents

Abstract

The present invention relates to a novel biomarker for diagnosing liver cancer and its use, and more particularly to an ERR? (Estrogen-related receptor gamma) as a biomarker for diagnosis of liver cancer and its use as a diagnostic tool for liver cancer. The novel liver cancer biomarker ERRγ, a kit for detecting liver cancer including the means for detecting the same, and a method for providing information for the diagnosis of liver cancer using the kit are compared with the conventional diagnosis using AFP, and the sensitivity and specificity It can be remarkably elevated so that it can be effectively applied to clinics.

Description

ERR? As liver cancer biomarker and its use {ERR? AS AS BIOMAKER TO LIVER CANCER AND USE THEREOF}

The present invention relates to a novel biomarker for diagnosing liver cancer and its use, and more particularly to an ERR? (Estrogen-related receptor gamma) as a biomarker for diagnosis of liver cancer and its use as a diagnostic tool for liver cancer.

Liver cancer is the sixth most common cancer worldwide and is the highest cause of cancer-related deaths. Although hepatic resection is the primary treatment for hepatocellular carcinoma, only 30-40% of patients initially diagnosed with hepatocellular carcinoma can undergo surgical treatment. Conservative chemotherapy for advanced hepatocellular carcinoma, which can not undergo surgery, Is becoming more important.

The sensitivity and specificity of high-risk patients were 39-97% and 76-95%, respectively, and the variance was significant and positive predictive value Is also low at 9 ~ 32%. In addition, it was not specifically increased in liver cancer, and there was a limitation in using it as an early diagnosis biomarker because it was normal in the early stage of liver cancer.

Recently, the expression of orphan nuclear receptor, which has not yet been identified in various cancers including liver cancer, affects the development and prognosis of cancer, and the cancer-specific expression of these orphan nuclear receptors is clinically evaluated Active research is being carried out.

KR 10-1200194 B KR 10-1161789 B KR 10-1004960 B

The present invention provides a new liver cancer biomarker, ERRγ, a kind of oropharyngeal nuclear receptor, a liver cancer diagnostic kit capable of detecting the biomarker, and a method for providing information for diagnosis of liver cancer using the kit.

In order to solve the above problems, the present invention provides a biomarker composition for diagnosing liver cancer comprising ERR ?.

The ERR? May be a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1.

The ERR? May be a protein having an amino acid sequence represented by SEQ ID NO: 2.

The present invention also provides a kit for the diagnosis of liver cancer, which comprises means capable of measuring the expression level of ERR ?.

The means is preferably selected from the group consisting of primers, probes, antibodies, antibody fragments, microarrays and aptamers.

The present invention also provides a method for providing information for the diagnosis of liver cancer, which comprises detecting the expression of ERR? From a biological sample.

The biological sample is preferably selected from the group consisting of liver tissue, liver cells, blood, serum, saliva and urine.

Preferably, the expression is mRNA expression or protein expression.

Preferably, the detection is detection of mRNA expression or detection of protein expression.

The mRNA expression may be detected by RT-PCR, northern blotting, or a nucleotide microarray.

The detection of the protein expression is preferably an antigen-antibody reaction.

The detection by the antigen-antibody reaction can be performed by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry or protein microarray or aptamer, It is preferable that the detection is performed by using

The novel liver cancer biomarker ERRγ, a kit for detecting liver cancer including the means for detecting the same, and a method for providing information for the diagnosis of liver cancer using the kit are compared with the conventional diagnosis using AFP, and the sensitivity and specificity It can be remarkably elevated so that it can be effectively applied to clinics.

FIG. 1 is a graph showing the expression patterns of ERRγ in normal liver and liver cancer tissues. FIG.

Hereinafter, the present invention will be described in detail.

Although AFP is the most commonly used tumor marker for hepatocellular carcinoma, the sensitivity and specificity of AFP in high risk group are 39% ~ 97%, 76 ~ 95%, and 9 ~ 32%, respectively. In addition, there was a limitation in using as a biomarker for the early diagnosis of liver cancer because it was not specifically increased in liver cancer and it was often normal in the early stage of liver cancer. If hepatocellular carcinoma is not diagnosed at an early stage, there is no clear treatment method. In addition to AFP, development of a biomarker that is helpful for early diagnosis was required.

The inventors of the present invention continued to develop a novel molecular marker for diagnosing early liver cancer. As a result, it was confirmed that the expression of human ERRγ was increased in liver cancer, but not expressed in normal liver tissue. Respectively. This suggests that the expression of ERRγ may be used as a new biomarker in the early diagnosis of liver cancer.

Accordingly, the present invention provides a biomarker composition for diagnosing liver cancer comprising ERR ?.

In the present invention, as described above, the expression of the oropharyngeal receptor ERR gamma is increased in liver cancer, while ERR gamma is not expressed in most normal liver tissues. In addition, the expression of ERRγ was significantly different from that of liver cancer stage, and ERRγ expression was increased with increasing stage. In particular, the patients registered in the present invention are early diagnosed liver cancer patients who can be operated on, and the possibility that the expression of ERR gamma may be used as an important diagnostic marker in early liver cancer patients is suggested.

ERRγ, an orphan nuclear receptor, is one of the ERR groups. In the present invention, the degree of expression of ERRγ was analyzed in 190 patients who underwent surgical liver resection for hepatocellular carcinoma. The sensitivity and specificity of ERRγ expression were 97.9% and 71%, respectively. The positive and negative predictive values were also 97.1% and 76.8%, respectively. AFP.

The biomarker composition of the present invention means a molecular marker for early diagnosis of liver cancer. ERR? Constituting the biomarker composition may be a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 or a protein having the amino acid sequence shown in SEQ ID NO: 2. Also, it may be any base sequence or amino acid sequence having a homology of 70%, preferably 80%, more preferably 90% or more, to the base sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2.

The present invention also provides a kit for the diagnosis of liver cancer, which comprises means capable of measuring the expression level of ERR ?.

The above-mentioned "ERR?" Has the same meaning as the content of the biomarker composition of the present invention described above.

The expression amount of ERR? Refers to the amount of ERR? MRNA expression or protein expression.

The means may be a known molecular biological means or immunochemical means.

In a preferred embodiment, the means may be a measuring means selected from the group consisting of primers, probes, antibodies, antibody fragments, microarrays and aptamers. Here, the primer, the probe, or the nucleic acid microarray corresponds to a means for measuring the expression amount of mRNA, and the antibody, the antibody fragment, the protein microarray or the aptamer corresponds to the means for measuring the expression amount of the protein.

The term "primer" refers to a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerization enzymes) within a suitable buffer and a suitable temperature it means. The appropriate length of the primer may vary depending on various factors, such as temperature and use of the primer. In addition, the sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer of the present invention does not need to have a perfectly complementary sequence to the nucleotide sequence of the ERR? Gene, which is the template, and it is sufficient that the primer has sufficient complementarity within the range capable of hybridizing to the gene sequence to perform the primer action.

The term "probe" means a linear oligomer of natural or modified monomers or linkages, which can specifically hybridize to a nucleotide sequence of a target nucleotide sequence of ERRy comprising deoxyribonucleotides and ribonucleotides, Or artificially synthesized. The probes according to the present invention may be single-stranded, preferably oligodeoxyribonucleotides. Probes of the invention can include natural dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), nucleotide analogs or derivatives. In addition, the probe of the present invention may also include a ribonucleotide. For example, the probes of the present invention can be used in combination with a framework-modified nucleotide such as a peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoamidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'- 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexy Tolyl DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines wherein the substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, 7-deazapurines having a C-7 substituent (the substituents being fluoro, bromo, chloro, bromo, bromo, -, Iodo-, methyl- , Ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-, inosine and diaminopurine.

The "antibody" may include a polyclonal antibody, a monoclonal antibody, a recombinant antibody, or the like capable of specifically binding to the ERR [gamma] protein.

The antibody is commercially available and can be used. Since the amino acid sequence of ERR? Protein is known as described above, the antibody can be directly prepared using the amino acid sequence.

For example, in the case of a polyclonal antibody, an ERR gamma antigen may be injected into an animal, and blood may be collected from an animal to produce an antibody, Such polyclonal antibodies can be prepared from any animal species host, such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, and the like, which are well known in the art for obtaining serum. Monoclonal antibodies can be prepared using the hybridoma method (Kohler et al., European Jounal of Immunology, 6, 511-519, 1976) well known in the art or can be prepared using the phage antibody library Clackson et al., Nature, 352, 624-628, 1991, Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991).

The term "antibody fragment" means a functional fragment of an antibody molecule. The functional fragment of an antibody molecule is an antibody fragment having at least an antigen binding function, for example, Fab, F (ab '), F ') 2 and Fv.

The "microarray" may be, for example, a nucleic acid microarray for detecting ERR gamma gene or a protein microarray for detecting ERR gamma protein.

In the microarray of the present invention, a primer, a probe or an antibody capable of measuring the expression level of the ERR? Protein or a gene encoding the ERR? Protein is used as a hybridizable array element and immobilized on a substrate. Preferred substrates may include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports have. The hybridization array elements are arranged and immobilized on the substrate, and such immobilization can be performed by chemical bonding methods or covalent bonding methods such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a polylysine coating surface. In addition, the hybridization array element can be coupled to the substrate via a linker (e.g., ethylene glycol oligomer and diamine).

The "aptamer" means a nucleic acid oligomer having affinity for the ERR gamma protein. The aptamers synthesize randomly synthesized DNA oligomers consisting of about 60 bases. Among them, a DNA oligomer that binds to the ERR? Protein is separated into an affinity column, and the amplification and the separation process are repeated to give affinity to ERR? Can be produced.

The kit for the diagnosis of liver cancer of the present invention may be used for ERR gamma gene detection or ERR gamma protein detection.

When the kit for diagnosing liver cancer of the present invention is for gene amplification to be applied to a PCR amplification process, the kit of the present invention may optionally contain a reagent necessary for PCR amplification, such as a buffer, a DNA polymerase (for example, Thermus aquaticus (Taq), Thermus thermophilus (Thermostable DNA polymerases obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs, and the liver cancer diagnostic kit of the present invention may be used for immunological assays For the detection of applied ERR [gamma] proteins, the kit of the present invention may optionally comprise a secondary antibody and a labeling substrate. Further, the kit according to the present invention may be manufactured from a number of separate packaging or compartments containing the reagent components described above.

The present invention also provides a method for providing information for the diagnosis of liver cancer, which comprises detecting the expression of ERR? From a biological sample.

The detection can be quantitatively determined by measuring the level of the ERR gamma gene or protein and can be measured by, for example, signal intensity levels (0.12 and 3) as shown in the following examples.

In the present invention, the "biological sample" refers to a sample collected from a living body different from the normal control group in the expression level of the ERR gamma gene or the expression level of the ERR gamma protein according to the degree of liver cancer development or progression. Liver tissue, liver cells, blood, serum, saliva, or urine.

The measurement of the expression level of the ERRγ gene preferably measures the level of mRNA, and in this case, a process of extracting RNA from the biological sample should be performed. RT-PCR, real-time RT-PCR, RNase protection assay, northern blotting (RT-PCR), and Northern blotting ) Or a nucleotide microarray, but are not limited thereto.

In this case, the ERR gamma marker protein in the biological sample and the antibody specific thereto form a conjugate, that is, an antigen-antibody complex, and the amount of the antigen- can be quantitatively measured through the magnitude of the signal of the detection label. Such detection labels may be selected from the group consisting of enzymes, chromophores, ligands, emitters, microparticles, redox molecules and radioactive isotopes, but is not limited thereto. Analysis methods for measuring protein levels include western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry or protein microarray, or aptamer aptamer), but the present invention is not limited thereto.

The level of the ERR gamma gene or protein can be quantitatively evaluated according to the measurement method as described above, and the basic information for the clinical judgment of the occurrence of liver cancer can be provided according to the expression level thereof.

Hereinafter, the present invention will be described in more detail with reference to concrete examples.

[ Example ]

1. Method

We retrospectively studied 190 patients who underwent liver cancer surgery at Keimyung University Dongsan Medical Center from 2001 to 2011. Patients who underwent hepatocellular chemoembolization or radiofrequency ablation to exclude unforeseen disturbance parameters were excluded from registration.

We analyzed the size of the tumors at the time of resection and analyzed the cause of liver cancer according to the TNM stage and Barcelona Clinic Liver Cancer (BCLC) Respectively.

Tissue microarray blocks were prepared from formalin - fixed, paraffin - embedded tissue tissues and normal tissues of peripheral liver cancer tissues. Immunohistochemical staining was performed using ERRγ antibody. The degree of staining was classified according to the staining rate of cancer cells (0, no staining; 1, weak; 2, moderate; 3, strong). Statistical significance was analyzed using the 18th SPSS based on clinical data.

2. result

Of the 190 patients who underwent hepatectomy, 158 (83%) were male, and 137 (72%) of all hepatocellular carcinomas were hepatocellular carcinoma due to HBV. There was no statistically significant difference in sex, age, etiology of liver cancer, and cirrhosis between ERRγ low (ERRγ0-1) group with low expression of ERRγ and ERRγ high (ERRγ 2-3) group with high expression of ERRγ Table 1).

[Table 1] 190 patients with liver cancer ERR γ expression and clinical features

However, when we analyzed the stage of liver cancer according to the level of expression of ERRγ, there were many groups with low expression of ERRγ in stage I and II according to the TNM staging system. In the case of stage III with cancer, the ratio of the group with high expression of ERRγ was significantly higher And it was found. Similar results were obtained when classified according to the BCLC stage (Fig. 1).

Finally, we analyzed the expression level of ERRγ in liver cancer tissues and surrounding normal liver tissues. Interestingly, ERRγ was not expressed in 96.3% of normal liver tissues, whereas ERRγ was expressed in 71% of liver cancer tissues. The specificity of ERRγ expression in liver cancer was 97.9% (183/183 + 4) and the sensitivity was 71% (135/135 + 55) , Positive predictive value and negative predictive value were also 97.1% (135/135 + 4) and 76.8% (183/183 + 55), respectively.

[Table 2] Normal Liver tissue  In liver cancer tissue ERR Expression analysis of?

<110> KYUNGPOOK NATIONAL UNIVERSITY HOSPITAL <120> ERR GAMMA AS THE BIOMAKER TO LIVER CANCER AND USE THEREOF <130> PP14-057 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1308 <212> DNA <213> Homo sapiens ERR-gamma coding sequence <400> 1 atgtcaaaca aagatcgaca cattgattcc agctgttcgt ccttcatcaa gacggaacct 60 tccagcccag cctccctgac ggacagcgtc aaccaccaca gccctggtgg ctcttcagac 120 gccagtggga gctacagttc aaccatgaat ggccatcaga acggacttga ctcgccacct 180 ctctaccctt ctgctcctat cctgggaggt agtgggcctg tcaggaaact gtatgatgac 240 tgctccagca ccattgttga agatccccag accaagtgtg aatacatgct caactcgatg 300 cccaagagac tgtgtttagt gtgtggtgac atcgcttctg ggtaccacta tggggtagca 360 tcatgtgaag cctgcaaggc attcttcaag aggacaattc aaggcaatat agaatacagc 420 tgccctgcca cgaatgaatg tgaaatcaca aagcgcagac gtaaatcctg ccaggcttgc 480 cgcttcatga agtgtttaaa agtgggcatg ctgaaagaag gggtgcgtct tgacagagta 540 cgtggaggtc ggcagaagta caagcgcagg atagatgcgg agaacagccc atacctgaac 600 cctcagctgg ttcagccagc caaaaagcca tataacaaga ttgtctcaca tttgttggtg 660 gctgaaccgg agaagatcta tgccatgcct gaccctactg tccccgacag tgacatcaaa 720 gccctcacta cactgtgtga cttggccgac cgagagttgg tggttatcat tggatgggcg 780 aagcatattc caggcttctc cacgctgtcc ctggcggacc agatgagcct tctgcagagt 840 gcttggatgg aaattttgat ccttggtgtc gtataccggt ctctttcgtt tgaggatgaa 900 cttgtctatg cagacgatta tataatggac gaagaccagt ccaaattagc aggccttctt 960 gatctaaata atgctatcct gcagctggta aagaaataca agagcatgaa gctggaaaaa 1020 gaagaatttg tcaccctcaa agctatagct cttgctaatt cagactccat gcacatagaa 1080 gatgttgaag ccgttcagaa gcttcaggat gtcttacatg aagcgctgca ggattatgaa 1140 gctggccagc acatggaaga ccctcgtcga gctggcaaga tgctgatgac actgccactc 1200 ctgaggcaga cctctaccaa ggccgtgcag catttctaca acatcaaact agaaggcaaa 1260 gtcccaatgc acaaactttt tttggaaatg ttggaggcca aggtctga 1308 <210> 2 <211> 435 <212> PRT <213> Homo sapiens ERR-gamma amino acid sequence <400> 2 Met Ser Asn Lys Asp Arg His Ile Asp Ser Ser Cys Ser Ser Phe Ile   1 5 10 15 Lys Thr Glu Pro Ser Ser Pro Ala Ser Leu Thr Asp Ser Val Asn His              20 25 30 His Ser Pro Gly Gly Ser Ser Asp Ala Ser Gly Ser Tyr Ser Ser Thr          35 40 45 Met Asn Gly His Gln Asn Gly Leu Asp Ser Pro Pro Leu Tyr Pro Ser      50 55 60 Ala Pro Ile Leu Gly Gly Ser Gly Pro Val Arg Lys Leu Tyr Asp Asp  65 70 75 80 Cys Ser Ser Thr Ile Val Glu Asp Pro Gln Thr Lys Cys Glu Tyr Met                  85 90 95 Leu Asn Ser Met Pro Lys Arg Leu Cys Leu Val Cys Gly Asp Ile Ala             100 105 110 Ser Gly Tyr His Tyr Gly Val Ala Ser Cys Glu Ala Cys Lys Ala Phe         115 120 125 Phe Lys Arg Thr Ile Gln Gly Asn Ile Glu Tyr Ser Cys Pro Ala Thr     130 135 140 Asn Glu Cys Glu Ile Thr Lys Arg Arg Arg Lys Ser Cys Gln Ala Cys 145 150 155 160 Arg Phe Met Lys Cys Leu Lys Val Gly Met Leu Lys Glu Gly Val Arg                 165 170 175 Leu Asp Arg Val Arg Gly Gly Arg Gln Lys Tyr Lys Arg Arg Ile Asp             180 185 190 Ala Glu Asn Ser Pro Tyr Leu Asn Pro Gln Leu Val Gln Pro Ala Lys         195 200 205 Lys Pro Tyr Asn Lys Ile Val Ser His Leu Leu Val Ala Glu Pro Glu     210 215 220 Lys Ile Tyr Ala Met Pro Asp Pro Thr Val Pro Asp Ser Asp Ile Lys 225 230 235 240 Ala Leu Thr Thr Leu Cys Asp Leu Ala Asp Arg Glu Leu Val Val Ile                 245 250 255 Ile Gly Trp Ala Lys His Ile Pro Gly Phe Ser Thr Leu Ser Leu Ala             260 265 270 Asp Gln Met Ser Leu Leu Gln Ser Ala Trp Met Glu Ile Leu Ile Leu         275 280 285 Gly Val Val Tyr Arg Ser Leu Ser Phe Glu Asp Glu Leu Val Tyr Ala     290 295 300 Asp Asp Tyr Ile Met Asp Glu Asp Gln Ser Lys Leu Ala Gly Leu Leu 305 310 315 320 Asp Leu Asn Asn Ale Ile Leu Gln Leu Val Lys Lys Tyr Lys Ser Met                 325 330 335 Lys Leu Glu Lys Glu Glu Phe Val Thr Leu Lys Ala Ile Ala Leu Ala             340 345 350 Asn Ser Asp Ser Met His Ile Glu Asp Val Glu Ala Val Gln Lys Leu         355 360 365 Gln Asp Val Leu His Glu Ala Leu Gln Asp Tyr Glu Ala Gly Gln His     370 375 380 Met Glu Asp Pro Arg Arg Ala Gly Lys Met Leu Met Thr Leu Pro Leu 385 390 395 400 Leu Arg Gln Thr Ser Thr Lys Ala Val Gln His Phe Tyr Asn Ile Lys                 405 410 415 Leu Glu Gly Lys Val Pro Met Lys Leu Phe Leu Glu Met Leu Glu             420 425 430 Ala Lys Val         435

Claims (12)

A biomarker composition for diagnosing liver cancer comprising ERR? (Estrogen-related receptor gamma) protein. delete [Claim 2] The biomarker composition for diagnosing liver cancer according to claim 1, wherein the ERR [gamma] protein is a protein having an amino acid sequence represented by SEQ ID NO: 2. A kit for the diagnosis of liver cancer, comprising means for measuring the expression level of ERR? (Estrogen-related receptor gamma) protein. The kit for diagnosing liver cancer according to claim 4, wherein the means is an antibody or an antibody fragment. A method for providing information for the diagnosis of liver cancer, which comprises detecting the expression of ERR? (Estrogen-related receptor gamma) protein from a biological sample. The method according to claim 6, wherein the biological sample is selected from the group consisting of liver tissue, liver cells, blood, serum, saliva, and urine. delete delete delete [Claim 7] The method according to claim 6, wherein the expression of the protein is detected by an antigen-antibody reaction. The method according to claim 11, wherein the detection by the antigen-antibody reaction is carried out by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry or protein microarray, Wherein the detection of the liver cancer is performed by the detection means.

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