KR101054951B1 - CAPN12, a molecule for diagnosing and treating liver cancer, and a kit comprising the same - Google Patents

Abstract

The present invention provides a marker detecting composition for diagnosing and treating liver cancer, including a preparation for measuring the expression level of CAPN12 (calpain 12), a kit comprising the composition, a microarray for diagnosing liver cancer using the marker, and It relates to a method for detecting the marker. According to the present invention, the marker may be utilized for early diagnosis of liver cancer to quickly determine the time of surgery or medication for liver cancer patients, and thus, may significantly contribute to improving survival.

Liver Cancer, Marker, Microarray, Diagnostics, Kit

Description

CAPN12, the molecule for diagnosing and treating liver cancer, the kit comprising the same {CAPN12, the molecular marker for diagnosing and curing hepatocellular carcinoma and a a kit, including the marker}

The present invention provides a marker detecting composition for diagnosing and treating liver cancer, including a preparation for measuring the expression level of CAPN12 (calpain 12), a kit comprising the composition, a microarray for diagnosing liver cancer using the marker, and It relates to a method for detecting the marker. According to the present invention, the marker may be utilized for early diagnosis of liver cancer to quickly determine the time of surgery or medication for liver cancer patients, and thus, may significantly contribute to improving survival.

Hepatocellular carcinoma is one of the most deadly cancers in the world, with more than half a million people dying from liver cancer each year in Asia and sub-Saharan Africa. In China, liver cancer is fatal, making it the second leading cause of death from cancer in China. Although the risk factor for liver cancer is chronic infection with hepatitis B or hepatitis C virus, the molecular mechanisms in liver cancer cells are still unclear. Recently, studies on markers for diagnosing liver cancer and predicting postoperative survival of patients have been steadily progressing, but diagnostic markers having excellent effects have not been developed. In addition, liver resection is a treatment for relatively early patients without metastases among liver cancer patients. Currently, about 20% of all liver cancer patients are treated by liver resection. Is not high. In particular, many patients die about 30-40% of survival within 5 years after surgery. In addition, a method for diagnosing liver cancer by clinical pathology has been used in the past, but it has not been applied to all liver cancer patients due to problems of universality and accuracy. Therefore, there is a great need to develop effective and specific liver cancer diagnostic markers.

Recent studies have shown that genes such as p53, beta-catenin, AXINI, p21 (WAF1 / CIP1) and p27 Kip are involved in the development of liver cancer. However, these individual gene changes do not accurately reflect the external and clinical characteristics of liver cancer patients, so the diversity of cells and molecules within individual liver cancers requires a new approach in addition to conventional genetic studies.

In this regard, microarray technology is a new technology that can simultaneously measure the expression of tens of thousands of genes in a single experiment beyond the range of individual genes, and has been applied to almost all cancer research including liver cancer. By extracting genes that actively participate in the process, the level of cancer diagnosis and prognosis is made possible at the molecular level. Although the diagnosis of liver cancer is difficult to determine accurately by their pathological findings, it is possible to distinguish liver cancer from non-tumor liver tissue by differences in their molecular expression profiles. This is because the difference in molecular expression profiles makes it possible to select the exact set of genes involved in liver cancer development. It is also expected to facilitate setting appropriate targets for treatment and thus maximize the effectiveness of treating liver cancer.

The present inventors endeavored to develop markers capable of accurately confirming liver cancer diagnosis at the molecular level through cDNA microarray analysis and quantitative reverse transcription PCR. As a result, tens of thousands of genes whose expression difference was largely expressed through microarray were reconfirmed through quantitative reverse transcription polymerase chain reaction (PCR), and the mRNA level was significantly higher in tumor tissue than in non-tumor tissue. The gene to be expressed could be confirmed. Accordingly, the present inventors have completed the present invention by identifying that CAPN12 is a liver cancer specific expression gene.

Accordingly, it is an object of the present invention to provide a composition for detecting markers for diagnosing liver cancer, comprising an agent for measuring the expression level of CAPN12.

Another object of the present invention is to provide a kit for detecting markers for diagnosing liver cancer, comprising the composition.

Still another object of the present invention is to provide a microarray for diagnosing liver cancer, including CAPN12.

Another object of the present invention is to provide a method for detecting liver cancer marker CAPN12.

As one aspect for achieving the above object, the present invention relates to a composition for detecting markers for diagnosing and treating liver cancer, comprising an agent for measuring the expression level of CAPN12 (calpain 12).

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether the liver cancer.

As used herein, the term "diagnostic marker, diagnostic marker, or diagnostic marker" is a substance capable of diagnosing liver cancer cells from normal cells, and increases or decreases in cells with liver cancer compared to normal cells. Organic polypeptides such as visible polypeptides or nucleic acids such as mRNA, lipids, glycolipids, glycoproteins, and the like. For purposes of the present invention, the hepatic cancer diagnostic markers of the present invention are CAPN12 (calpain 12) genes and proteins encoded thereby, which show a particularly high level of expression in liver cancer cells as compared to normal liver tissue cells.

CAPN12 (calpain 12) belongs to the family of cytosolic calcium-activated cysteine proteases and is known to be involved in cellular processes such as apoptosis and cell division. CAPN12 (calpain 12) gene and its protein information are registered in the National Center for Biotechnology Information (NCBI) (GeneID: 147968, NP_653292.2). Herein, the gene and amino acid sequences of CAPN12 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Moreover, the specific function of CAPN12 in liver cancer and the association with liver cancer are not known at all.

The present inventors have found that CAPN12 can be a diagnostic marker for liver cancer through the following verification.

Specifically, RNA was isolated and purified from 146 tumor tissues from liver cancer patients and 5 normal tissues from non-liver cancer patients. RNA purified from five normal tissues was mixed and used as a control RNA. Thus, cDNA was prepared by reverse transcription of RNA and control RNA isolated from tissues. In the process, Cy5-dUTP and Cy3-dUTP were bound to cDNA, respectively. This hybridized DNA chip was measured the intensity (intensity) of Cy5 and Cy3 at each spot using a laser scanner (Fig. 1). The relative ratios of these two types of fluorescence intensities were quantified using computer software (IMAGENE program) and the expression levels were measured. Numerical expression was normalized to select only genes with a survival correlation (p) of 0.05 or less, and overexpressed in tumors of 1.5fold or more with average 30% intensity averages of 1000 or less in each tissue in normal tissues. Only genes with more than 40 samples were selected. Subsequently, after subtracting the average value of the top 30% intensity of each gene expression from normal tissue from the top 30% intensity average of each gene expression in tumor tissue (146), genes with an IQR of 0.43 or higher, representing a difference in expression level in one gene, 18 The dog was finally selected. Genes selected through this process were reconfirmed through RT-PCR and agarose electrophoresis in 30 pairs of tumor and non-tumor tissues (FIG. 4).

In the present invention, the term "agent for measuring the expression level of CAPN12" refers to a molecule that can be used for detection of a marker by confirming the expression level of CAPN12, a marker that increases expression in liver cancer cells as described above. Refers to antibodies, primers or probes specific for the marker.

The expression level of CAPN12 can be determined by confirming the mRNA expression level of the CAPN12 gene or the expression level of the protein encoded by the gene. In the present invention, "mRNA expression level measurement" refers to a process of confirming the presence and expression level of mRNA of liver cancer marker gene in a biological sample in order to diagnose liver cancer, by measuring the amount of mRNA. Analytical methods for this include RT-PCR, competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), northern blotting (northern) blotting), DNA chips, and the like, but is not limited thereto.

In the present invention, "protein expression level measurement" refers to a process of confirming the presence and expression level of a protein expressed in a liver cancer marker gene in a biological sample to diagnose liver cancer. An antibody that specifically binds to a protein of the gene Check the amount of protein using. Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, and Rocket immunoelectrolysis There are, but are not limited to, electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like.

Agents for measuring mRNA levels of genes are preferably primer pairs or probes, and since the nucleic acid sequence of the CAPN12 gene is identified in NM_144691 (NCBI), those skilled in the art will appreciate primers that specifically amplify specific regions of these genes based on the sequence. Alternatively, the probe can be designed.

As used herein, the term “primer” refers to a nucleic acid sequence having a short free 3 ′ hydroxyl group, which may form complementary templates and base pairs and is a starting point for copying a template. By short nucleic acid sequence is meant to function. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. In the present invention, PCR can be performed using the sense and antisense primers of the CAPN12 polynucleotide to diagnose liver cancer through the generation of a desired product. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.

 As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which is short to several bases to hundreds of bases, which is capable of specific binding with mRNA. You can check the presence. Probes may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, and the like. In the present invention, hybridization may be performed using a probe complementary to a CAPN12 polynucleotide, and liver cancer may be diagnosed through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosph Modifications to poroamidates, carbamates, and the like) or charged linkers (eg, phosphorothioates, phosphorodithioates, etc.).

Agents for measuring protein levels are preferably antibodies.

As used herein, the term "antibody" refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody means an antibody that specifically binds to CAPN12, a marker of the present invention, which is a protein encoded by the marker gene by cloning each gene into an expression vector according to a conventional method. Can be obtained and prepared from conventional proteins by conventional methods. Also included are partial peptides that may be made from such proteins, and the partial peptides of the present invention include at least seven amino acids, preferably nine amino acids, more preferably twelve or more amino acids.

The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.

Antibodies used in the detection of liver cancer diagnostic markers of the invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. The functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, and Fv.

In another aspect, the present invention relates to a marker detection kit for diagnosing liver cancer, comprising the composition for detecting liver cancer diagnostic marker.

The kit of the present invention can detect the marker by checking the mRNA expression level or protein expression level of CAPN12, a liver cancer diagnosis marker. The marker detection kit of the present invention includes a primer, a probe, or an antibody that selectively recognizes a marker for measuring the expression level of a liver cancer diagnostic marker, as well as one or more other component compositions, solutions, or devices suitable for analytical methods. May be included.

As a specific example, the kit for measuring the mRNA expression level of CAPN12 in the present invention may be a kit containing the necessary elements necessary to perform RT-PCR. RT-PCR kits, in addition to each primer pair specific for the marker gene, RT-PCR kits can be used in test tubes or other appropriate containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerases and reverse transcriptases, DNase, RNase inhibitors, DEPC-water (DEPC-water), sterile water, and the like. In addition, the kit of the present invention may be a kit for detecting a diagnostic marker including an essential element necessary to perform a DNA chip. The DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached with a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.

As another specific example, the kit for measuring the protein expression level of CAPN12 in the present invention comprises a substrate, a suitable buffer, a secondary antibody labeled with a chromophore or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. It may include. The substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like. The chromophore may be a peroxidase or an alkaline force. Fatase (alkaline phosphatase) can be used, and fluorescent materials can be used, such as FITC, RITC, and the colorant substrate is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6- Sulfonic acid)) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.

As another aspect, the invention relates to a microarray for diagnosing liver cancer, comprising a marker according to the invention. The microarray of the present invention can be easily prepared by a manufacturing method commonly used in the art using the marker of the present invention.

In another aspect, the present invention relates to a method for detecting liver cancer marker CAPN12 by comparing the expression level of CAPN12 from a patient's sample with the expression level of normal tissue to provide information necessary for diagnosing liver cancer.

More specifically, expression of genes can be detected at the mRNA level or at the protein level, and the separation of mRNA or protein from biological samples can be performed using known processes.

As used herein, the term "sample of patient" includes, but is not limited to, a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, which differ in the expression level of the liver cancer marker gene CAPN12. .

Through the detection methods, it is possible to diagnose whether the cancer patient is a real cancer patient by comparing the gene expression level in the normal control group with the gene expression level in the suspected liver cancer patient. That is, after measuring the expression level of the marker of the present invention from the cells suspected of liver cancer, and comparing the two by measuring the expression level of the marker of the present invention from normal cells, the expression level of the marker of the present invention is that of normal cells. If more expression is derived from a cell suspected of liver cancer, a cell suspected of liver cancer can be predicted as liver cancer.

Analytical methods for measuring mRNA 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 mRNA expression level in the normal control group and the mRNA expression level in the suspected liver cancer patient, and to determine whether the liver cancer marker gene is significantly increased or decreased in the expression level of the liver cancer suspected patient. Can be diagnosed.

mRNA expression level measurement is preferably using a reverse transcriptase polymerase reaction method or a DNA chip using a primer specific for the gene used as a liver cancer marker.

The reverse transcriptase polymerase reaction is electrophoresis after the reaction to confirm the band pattern and the thickness of the band by checking the mRNA expression and degree of the gene used as a diagnostic marker for liver cancer, and compared with the control group, it is easy to determine the occurrence of liver cancer Diagnosis can be made.

On the other hand, the DNA chip is a DNA chip in which the nucleic acid corresponding to the liver cancer marker gene or a fragment thereof is attached to a glass-like substrate at a high density, and isolates the mRNA from the sample, cDNA labeled at the end or the inside of the fluorescent material Probes can be prepared, hybridized to DNA chips and read for the development of liver cancer.

Analytical methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, Protein chips and the like, but is not limited thereto. Through the above analysis methods, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in suspected liver cancer patients can be compared, and whether the significant expression level of liver cancer marker gene to protein is increased. By judging, it is possible to diagnose whether liver cancer suspected patient actually develops liver cancer.

As used herein, the term “antigen-antibody complex” means a combination of a liver cancer marker protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through the size of a signal of a detection label. Do.

Protein expression level measurement is preferably by using an ELISA method. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support Various ELISA methods include indirect sandwich ELISA using secondary antibodies. More preferably, the antibody is enzymatically developed by attaching the antibody to the solid support, reacting the sample, and then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It is detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops. By confirming the degree of complex formation between the liver cancer marker protein and the antibody, it is possible to determine whether the liver cancer.

Also preferably, Western blot using at least one antibody against the liver cancer marker. The whole protein is isolated from the sample, electrophoresed to separate the protein according to size, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the generated antigen-antibody complex can be confirmed by using a labeled antibody to confirm the amount of the protein produced by the expression of the gene, thereby confirming the occurrence of liver cancer. The detection method consists of a method of examining the expression level of the marker gene in the control group and the expression level of the marker gene in cells with liver cancer. mRNA or protein levels can be expressed as absolute (eg μg / ml) or relative (eg signal relative strength) of the marker proteins described above.

Also preferably, immunohistostaining is performed using at least one antibody against the liver cancer marker. After collecting and fixing normal colon epithelial tissue and tissue suspected of liver cancer, paraffin embedding blocks are prepared by methods well known in the art. These are cut into slices of several μm thickness, adhered to glass slides, and reacted with a selected one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.

Also, preferably, at least one antibody against the liver cancer marker is arranged at a predetermined position on the substrate to use a protein chip immobilized at a high density. In the method of analyzing a sample using a protein chip, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex, which is read to confirm the presence or expression level of the protein, You can check whether you have liver cancer.

In another aspect, the present invention provides a method for detecting liver cancer marker CAPN12 by comparing the expression level of CAPN12 with the expression level of normal cells from a sample of a patient undergoing liver cancer resection to provide information necessary for predicting the prognosis of liver cancer resection. It is about a method.

Through the detection method, it is possible to predict the prognosis of the patient who has undergone a new liver cancer resection surgery by analyzing the expression pattern of the predictive gene, and based on this prediction, it may be used for the prognosis treatment of the patient.

The present inventors identified and selected genes whose expression is remarkably increased in liver cancer tissues compared to normal liver tissues, and evaluated their applicability as diagnostic markers. Therefore, using the liver cancer diagnostic marker CAPN12 according to the present invention can accurately and easily measure the presence or absence of liver cancer, it can be further utilized in the study of tumor formation of liver cancer. It is also expected to contribute to the early diagnosis of liver cancer and the decision of timing of surgery and treatment.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example  One: Test group  And selection of test tissue samples

Test specimens were obtained from 98 patients who underwent surgery for liver cancer at Nuclear Hospital and 58 patients who underwent surgery for liver cancer at Seoul National University Hospital. Patients were informed to use surgical specimens and clinicopathological data for research purposes. All tissues were surgically extracted and immediately cooled in liquid nitrogen and stored at minus 80 ° C. RNA was isolated from cooled tissue according to the test manual (RNeasy MiniElute Cleanup Kit, Qiagen). The quality of total RNA was determined from the band ratio between 28S and 18S rRNA after agarose gel electrophoresis.

Example  2: cDNA Microarray  Data Analysis and Gene Screening

The microarrays used in the experiment consisted of a total of 24,094 human cDNA clones. Microarray experiments were performed using the 3DNA array detection kit (Genisphere Inc. PA, USA) according to the method suggested by the manufacturer. It was then scanned using a scan array scanner (PerkinElmer, Boston, Mass., USA). Numerical data were obtained from the scanned image file using IMAGENE 4.0 (Biodiescovery, Marina del Rey, Calif., USA), and normalized in consideration of fluorescence intensity and spot position (spatially dependent method). In the normalized microarray data, only those genes with a survival correlation (p) of 0.05 or less were selected from the normalization process, and the average 30% intensity of each gene expression in normal tissues was 1000 or less and 1.5 fold or more. Only genes with over 40 samples overexpressed in the tumor were selected. Subsequently, after subtracting the average value of the top 30% intensity of each gene expression from normal tissue from the top 30% intensity average of each gene expression in tumor tissue (146), genes with an IQR of 0.43 or higher, representing a difference in expression level in one gene, 18 The dog was finally selected. In addition, genes corresponding to liver cancer specific proteins, which are reported to have similarities with proteins released out of cells, were selected for more effective diagnostic methods, such as the easy measurement of liver cancer in the blood of patients.

Example  3: for predictive genes RT - PCR Expression reconfirmation and liver cancer prediction

In microarray data, CAPN12 was expressed more than 1.5fold in 97 tumor tissues compared to normal tissues among 146 tumor tissues. To reconfirm the expression of these genes, RNA was extracted from 30 tumor tissues, adjacent non-tumor tissues, and normal liver tissues of 5 patients, respectively, and cDNA was synthesized by reverse transcription. This was again confirmed CAPN12 gene expression through reverse transcriptase polymerase chain reaction (RT-PCR) under the following conditions (Fig. 3).

name Primer sequence (5 '→ 3' ) RT - PCR in
annealing Tm RT - PCR in
cycle ( cycle )Number F gccacatgaatgaggctttt
(SEQ ID NO: 3)
56 ℃
34 R ccaggaacaccttgtgtgtg
(SEQ ID NO: 4)

As a result, the expression of CAPN12 was increased in 23 tumor tissues compared with non-tumor tissues, and the expression of this marker was significantly increased in 30 tumor tissues compared to normal liver tissues.

Figure 1 is a schematic diagram showing the hybridization and analysis of the reverse transcription of mRNA and DNA chip for measuring the expression profile of liver cancer diagnostic gene.

Figure 2 shows the gene sequence and protein sequence of the selected liver cancer gene CAPN12.

Figure 3 shows the primer sequence for RT-PCR, which can measure the expression of the gene of CAPN12, the selected liver cancer diagnostic gene, the conditions of the RT-PCR experiment and the conserved domain.

Figure 4 is reconfirmed the expression of the selected liver cancer specific gene CAPN12 via RT-PCR in 30 tumor tissues.

<110> Korea Institute of Radiological and Medical Sciences <120> CAPN12, the molecular marker for diagnosing and curing          hepatocellular carcinoma and a kit, including the marker <130> PA090023 / KR <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 2160 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (2160) 223 nucleotide sequence of CAPN12 (calpain 12) <400> 1 atggcatcca gcagtgggag ggtcaccatc cagctcgtgg atgaggaggc tggggtcgga 60 gccgggcgcc tgcagctttt tcggggccag agctatgagg caattcgggc agcctgcctg 120 gattcgggga tcctgttccg cgacccttac ttccctgctg gccctgatgc ccttggctat 180 gaccagctgg ggccggactc ggagaaggcc aaaggcgtga aatggatgag gccccatgag 240 ttctgtgctg agccgaagtt catctgtgaa gacatgagcc gcacagacgt gtgtcagggg 300 agcctgggta actgctggtt ccttgcagcc gccgcctccc ttactctgta tccccggctc 360 ctgcgccggg tggtccctcc tggacaggat ttccagcatg gctacgcagg cgtcttccac 420 ttccagctct ggcagtttgg ccgctggatg gacgtcgtgg tggatgacag gctgcccgtg 480 cgtgagggga agctgatgtt cgtgcgctcg gaacagcgga atgagttctg ggccccactc 540 ctggagaagg cctacgccaa gctccacggc tcctatgagg tgatgcgggg cggccacatg 600 aatgaggctt ttgtggattt cacaggcggc gtgggcgagg tgctctatct gagacaaaac 660 agcatggggc tgttctctgc cctgcgccat gccctggcca aggagtccct cgtgggcgcc 720 actgccctga gtgatcgggg tgagtaccgc acagaagagg gcctggtaaa gggacacgcg 780 tattccatca cgggcacaca caaggtgttc ctgggcttca ccaaggtgcg gctgctgcgg 840 ctgcggaacc catggggctg cgtggagtgg acgggggcct ggagcgacag ctgcccacgc 900 tgggacacac tccccaccga gtgccgcgat gccctgctgg tgaaaaagga ggatggcgag 960 ttctggatgg agctgcggga cttcctcctc catttcgaca ccgtgcagat ctgctcgctg 1020 agcccggagg tgctgggccc cagcccggag gggggcggct ggcacgtcca caccttccaa 1080 ggccgctggg tgcgtggctt caactccggc gggagccagc ctaatgctga aaccttctgg 1140 accaatcctc agttccgttt aacgctgctg gagcctgatg aggaggatga cgaggatgag 1200 gaagggccct gggggggctg gggggctgca ggggcacggg gcccagcgcg ggggggccgc 1260 acgcccaagt gcacggtcct tctgtccctc atccagcgca accggcggcg cctgagagcc 1320 aagggcctca cttacctcac cgttggcttc cacgtgttcc agattccaga ggagctgctg 1380 ggcctctggg attccccgcg cagccatgcg ctcctgcccc ggctgctgcg cgccgaccgc 1440 tcgcccctca gcgcccgccg cgacgtgacc cgccgctgct gcctgcgtcc aggccactac 1500 ctggtggtgc cgagcaccgc ccacgccggc gacgaggctg acttcactct gcgtgtcttc 1560 tccgagcgcc gccacacggc cgtggagatc gacgacgtga tcagcgcaga cctgcagtct 1620 ctccagggcc cctacctgcc cctggagctg gggttggagc agctgtttca ggagctggct 1680 ggagaggagg aagaactcaa tgcctctcag ctccaggcct tactaagcat tgccctggag 1740 cctgccaggg cccatacctc cacccccaga gagatcgggc tcaggacctg tgagcagctg 1800 ctgcagtgtt tcgggcatgg gcaaagcctg gccttacacc acttccagca gctctggggc 1860 tacctcctgg agtggcaggc catatttaac aagttcgatg aggacacctc tggaaccatg 1920 aactcctacg agctgaggct ggcactgaat gcagcaggct tccacctgaa caaccagctg 1980 acccagaccc tcaccagccg ctaccgggat agccgtctgc gtgtggactt cgagcggttc 2040 gtgtcctgtg tggcccacct cacctgcatc ttctgccact gcagccagca cctggatggg 2100 ggtgaggggg tcatctgcct gacccacaga cagtggatgg aggtggccac cttctcctag 2160                                                                         2160 <210> 2 <211> 719 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE (222) (1) .. (719) <223> amino acid sequence of CAPN12 <400> 2 Met Ala Ser Ser Ser Gly Arg Val Thr Ile Gln Leu Val Asp Glu Glu   1 5 10 15 Ala Gly Val Gly Ala Gly Arg Leu Gln Leu Phe Arg Gly Gln Ser Tyr              20 25 30 Glu Ala Ile Arg Ala Ala Cys Leu Asp Ser Gly Ile Leu Phe Arg Asp          35 40 45 Pro Tyr Phe Pro Ala Gly Pro Asp Ala Leu Gly Tyr Asp Gln Leu Gly      50 55 60 Pro Asp Ser Glu Lys Ala Lys Gly Val Lys Trp Met Arg Pro His Glu  65 70 75 80 Phe Cys Ala Glu Pro Lys Phe Ile Cys Glu Asp Met Ser Arg Thr Asp                  85 90 95 Val Cys Gln Gly Ser Leu Gly Asn Cys Trp Phe Leu Ala Ala Ala Ala             100 105 110 Ser Leu Thr Leu Tyr Pro Arg Leu Leu Arg Arg Val Val Pro Pro Gly         115 120 125 Gln Asp Phe Gln His Gly Tyr Ala Gly Val Phe His Phe Gln Leu Trp     130 135 140 Gln Phe Gly Arg Trp Met Asp Val Val Val Asp Asp Arg Leu Pro Val 145 150 155 160 Arg Glu Gly Lys Leu Met Phe Val Arg Ser Glu Gln Arg Asn Glu Phe                 165 170 175 Trp Ala Pro Leu Leu Glu Lys Ala Tyr Ala Lys Leu His Gly Ser Tyr             180 185 190 Glu Val Met Arg Gly Gly His Met Asn Glu Ala Phe Val Asp Phe Thr         195 200 205 Gly Gly Val Gly Glu Val Leu Tyr Leu Arg Gln Asn Ser Met Gly Leu     210 215 220 Phe Ser Ala Leu Arg His Ala Leu Ala Lys Glu Ser Leu Val Gly Ala 225 230 235 240 Thr Ala Leu Ser Asp Arg Gly Glu Tyr Arg Thr Glu Glu Gly Leu Val                 245 250 255 Lys Gly His Ala Tyr Ser Ile Thr Gly Thr His Lys Val Phe Leu Gly             260 265 270 Phe Thr Lys Val Arg Leu Leu Arg Leu Arg Asn Pro Trp Gly Cys Val         275 280 285 Glu Trp Thr Gly Ala Trp Ser Asp Ser Cys Pro Arg Trp Asp Thr Leu     290 295 300 Pro Thr Glu Cys Arg Asp Ala Leu Leu Val Lys Lys Glu Asp Gly Glu 305 310 315 320 Phe Trp Met Glu Leu Arg Asp Phe Leu Leu His Phe Asp Thr Val Gln                 325 330 335 Ile Cys Ser Leu Ser Pro Glu Val Leu Gly Pro Ser Pro Glu Gly Gly             340 345 350 Gly Trp His Val His Thr Phe Gln Gly Arg Trp Val Arg Gly Phe Asn         355 360 365 Ser Gly Gly Ser Gln Pro Asn Ala Glu Thr Phe Trp Thr Asn Pro Gln     370 375 380 Phe Arg Leu Thr Leu Leu Glu Pro Asp Glu Glu Asp Asp Glu Asp Glu 385 390 395 400 Glu Gly Pro Trp Gly Gly Trp Gly Ala Ala Gly Ala Arg Gly Pro Ala                 405 410 415 Arg Gly Gly Arg Thr Pro Lys Cys Thr Val Leu Leu Ser Leu Ile Gln             420 425 430 Arg Asn Arg Arg Arg Leu Arg Ala Lys Gly Leu Thr Tyr Leu Thr Val         435 440 445 Gly Phe His Val Phe Gln Ile Pro Glu Glu Leu Leu Gly Leu Trp Asp     450 455 460 Ser Pro Arg Ser His Ala Leu Leu Pro Arg Leu Leu Arg Ala Asp Arg 465 470 475 480 Ser Pro Leu Ser Ala Arg Arg Asp Val Thr Arg Arg Cys Cys Leu Arg                 485 490 495 Pro Gly His Tyr Leu Val Val Pro Ser Thr Ala His Ala Gly Asp Glu             500 505 510 Ala Asp Phe Thr Leu Arg Val Phe Ser Glu Arg Arg His Thr Ala Val         515 520 525 Glu Ile Asp Asp Val Ile Ser Ala Asp Leu Gln Ser Leu Gln Gly Pro     530 535 540 Tyr Leu Pro Leu Glu Leu Gly Leu Glu Gln Leu Phe Gln Glu Leu Ala 545 550 555 560 Gly Glu Glu Glu Glu Leu Asn Ala Ser Gln Leu Gln Ala Leu Leu Ser                 565 570 575 Ile Ala Leu Glu Pro Ala Arg Ala His Thr Ser Thr Pro Arg Glu Ile             580 585 590 Gly Leu Arg Thr Cys Glu Gln Leu Leu Gln Cys Phe Gly His Gly Gln         595 600 605 Ser Leu Ala Leu His His Phe Gln Gln Leu Trp Gly Tyr Leu Leu Glu     610 615 620 Trp Gln Ala Ile Phe Asn Lys Phe Asp Glu Asp Thr Ser Gly Thr Met 625 630 635 640 Asn Ser Tyr Glu Leu Arg Leu Ala Leu Asn Ala Ala Gly Phe His Leu                 645 650 655 Asn Asn Gln Leu Thr Gln Thr Leu Thr Ser Arg Tyr Arg Asp Ser Arg             660 665 670 Leu Arg Val Asp Phe Glu Arg Phe Val Ser Cys Val Ala His Leu Thr         675 680 685 Cys Ile Phe Cys His Cys Ser Gln His Leu Asp Gly Gly Glu Gly Val     690 695 700 Ile Cys Leu Thr His Arg Gln Trp Met Glu Val Ala Thr Phe Ser 705 710 715 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for CAPN12 <400> 3 gccacatgaa tgaggctttt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for CAPN12 <400> 4 ccaggaacac cttgtgtgtg 20  

Claims (13)

A composition for detecting markers for diagnosing liver cancer, comprising an agent for measuring the expression level of mRNA of CAPN12 (calpain 12) gene or protein thereof. The composition of claim 1, wherein the agent for measuring the expression level of the gene mRNA is a primer pair that specifically binds to the CAPN12 gene. The composition of claim 1, wherein the agent for measuring the expression level of the gene mRNA is a probe that specifically binds to the CAPN12 gene. The composition of claim 1, wherein the agent measuring the expression level of the protein is an antibody specific for CAPN12 protein. Kit for diagnosing liver cancer comprising the composition of any one of claims 1 to 4. According to claim 5, The kit is liver cancer diagnostic kit, characterized in that the RT-PCR kit, DNA chip kit or protein chip kit. delete Measuring the mRNA expression level of the CAPN12 (calpain 12) gene or the protein encoded by the gene to provide information necessary for diagnosing liver cancer; Comparing the mRNA expression level of the gene or the level of the protein encoded by the gene with the expression level or protein level of the gene of the normal control sample; And judging liver cancer when the mRNA expression level of the patient sample is higher than that of the normal control sample. The method of claim 8, wherein the method of measuring mRNA expression level uses a primer pair or probe that specifically binds to the CAPN12 gene. The method of claim 8, wherein the mRNA level is measured using any one of reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip. How. The method of claim 8, wherein the protein expression level uses an antibody specific for that protein. The method of claim 8, wherein the method for measuring protein expression level is Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement Any one of a fixed assay, FACS or protein chip method. Comparing the expression level of CAPN12 (calpain 12) with the expression level of normal cells from a sample of patients undergoing liver cancer resection to provide information necessary for predicting the prognosis of liver cancer resection; And if the expression level of CAPN12 in the patient sample is not higher than the expression level of normal cells, determining the prognosis of liver cancer resection surgery is good. 13. The method of detecting liver cancer marker CAPN12 (calpain 12).

Images