KR101755792B1 - Biomarker for diagnosing or prognosing cancer and uses thereof - Google Patents

Abstract

The present invention relates to a composition, kit and method for diagnosing cancer or estimating prognosis by detecting the expression level of TIA-1 (T-cell restricted intracellular antigen 1) or MFF (mitochondrial fission factor) from a biological sample.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biomarker for cancer diagnosis or prognosis estimation,

The present invention relates to a composition, kit and method for diagnosing cancer or estimating prognosis by detecting the expression level of TIA-1 (T-cell restricted intracellular antigen 1) or MFF (mitochondrial fission factor) from a biological sample.

Cancer is one of the major causes of death globally. Symptoms often occur only after the disease progresses considerably, so that the appropriate treatment period is often missed, and many cases of cancer death are due to cancer recurrence. In addition, the prognosis of cancer refers to the prediction of the various conditions of the patient such as the possibility of cancer cure after cancer diagnosis, possibility of recurrence after treatment, survival possibility of the patient, and the like, And so on.

Therefore, not only can early treatment be possible by more accurately determining the onset and progression stage of cancer, but also to better determine the appropriate treatment method by predicting the clinical outcome of the patient after treatment, or to increase the survival rate after onset of cancer Development of biomarkers is required.

Korean Patent Publication No. 10-2010-0115283 (Oct. 27, 2010) Korean Patent Publication No. 10-2010-0067031 (June 18, 2010) Korean Patent Laid-Open No. 10-2014-0115490 (Apr. 201, 2014)

The present invention seeks to provide the use of TIA-1 and / or MFF as cancer diagnostic and prognostic markers.

Accordingly, one object of the present invention is to provide a composition for cancer diagnosis or prognosis estimation comprising a substance that measures the expression level of TIA-1 gene or MFF gene.

Another object of the present invention is to provide a kit for cancer diagnosis or prognosis estimation comprising the composition for cancer diagnosis or prognosis estimation.

Another object of the present invention is to provide a method for providing information for cancer diagnosis or prognosis estimation using the composition for cancer diagnosis or prognosis estimation.

It is another object of the present invention to provide a method for detecting a cancer marker for diagnosis or prognosis, comprising the step of measuring the expression level of TIA-1 gene or MFF gene present in a biological sample.

In order to achieve the above object, the present invention provides a composition for cancer diagnosis or prognosis estimation comprising a substance which measures the expression level of TIA-1 gene or MFF gene.

According to one embodiment of the present invention, the substance for measuring the expression level of the gene is one that detects at least one of the presence, abundance, and presence pattern of the mRNA transcribed by the gene, the protein encoded by the gene, .

According to an embodiment of the present invention, the substance for measuring the expression level of the gene may be specifically bound to any one or more selected from the group consisting of a nucleotide sequence of the gene, a complementary sequence thereof, a fragment of the nucleotide and a complementary sequence thereof A primer, a probe, an aptamer, and an antisense.

According to an embodiment of the present invention, the substance for measuring the expression level of the gene may be any one or more of a polypeptide encoded by the nucleotide sequence, a polypeptide encoded by the complementary sequence, and a polypeptide encoded by a fragment of the nucleotide sequence An antibody fragment, a ligand, a peptide nucleic acid (PNA), an aptamer, an avidity multimer, or an oligonucleotide that specifically binds to an antigen, And peptidomimetics. The term " peptidomimetics "

According to an embodiment of the present invention, the substance for measuring the level of expression of the gene may be selected from the group consisting of the presence or absence of a combination of a protein encoded by the TIA-1 gene or the MFF gene and a messenger RNA (mRNA) And the like.

According to an embodiment of the present invention, the substance for measuring the expression level of the gene may be selected from the group consisting of a reverse transcription polymerase chain reaction, a competitive polymerase chain reaction, a real-time polymerase chain reaction, an Nuclease protection assay (RNase, S1 nuclease assay) , DNA microarray method, Northern blot, Western blot, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry (Mass spectrometry, and protein microarray. The present invention is a detection reagent for measuring gene expression using any one or more of the following methods.

According to an example of the present invention, the cancer may be liver cancer, lung cancer or blood cancer.

The present invention provides a kit for cancer diagnosis or prognosis estimation comprising the above-described composition for cancer diagnosis or prognosis estimation.

According to an embodiment of the present invention, the kit is characterized in that the kit is any one or more selected from a microarray, a gene amplification kit, an immunoassay kit, a luminex assay kit, a protein microarray kit, and an ELISA kit.

The present invention provides a method for estimating cancer or prognosis, comprising the steps of: treating a biological sample with the composition for cancer diagnosis or prognosis as described above; Measuring the expression level of the TIA-1 gene or the MFF gene from the biological sample; And comparing the gene expression level measurement result with a reference value.

According to an embodiment of the present invention, the expression level of a gene in the cancer diagnosis or prognosis estimation method can be measured by a reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time PCR, Nuclease protection assay (RNase, S1 nuclease assay) , in situ hybridization method, DNA microarray method, Northern blot, Western blot, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay, complement fixation assay, FACS , Mass spectrometry, and protein microarray method.

The present invention relates to a method for detecting a cancer diagnosis or a biomarker for prognosis estimation by measuring the expression level of TIA-1 gene or MFF gene in a biological sample of a human in order to provide information necessary for diagnosis or prognosis estimation of cancer .

According to one embodiment of the present invention, the expression level of a gene in the method for detecting a biomarker for cancer diagnosis or prognosis estimation is determined by a method selected from the group consisting of a reverse transcription polymerase chain reaction, a competitive polymerase chain reaction, a real-time polymerase chain reaction, RNase, S1 nuclease assay, in situ hybridization, DNA microarray, Northern blot, Western blot, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay , Complement fixation assay, FACS, mass spectrometry, and protein microarray.

The biomarker provided by the present invention not only enables early treatment by more accurately determining the onset or progression of cancer, but also allows a better prediction of the clinical result of the patient after treatment to determine an appropriate treatment method, It can be used to increase the survival rate after onset.

FIG. 1 (a) is a graph showing the relative expression levels of TIA-1 mRNA in normal liver tissue, hepatocellular carcinoma (HCC), and hepatocellular carcinoma tissues (TG1, TG2 and TG3). (b) is a graph showing the relative expression level of TIA-1 mRNA in normal liver tissue (HCC) and normal liver tissue (Normal Liver). GSE45436 means the registration number of the Gene Expression Omnibus (GEO) database.
FIG. 2 shows Western blot analysis of expression of TIA-1 protein in normal liver tissues and liver cancer tissue samples isolated from nine patients, respectively. Numbers 5, 8, 14, 48, 54, 72, 22, 30 and 77 mean the number of the sample.
FIG. 3 shows Western blot analysis of MFF protein expression in normal liver tissues and liver cancer tissue samples isolated from nine patients, respectively. Numbers 5, 8, 14, 48, 54, 72, 22, 30 and 77 mean the number of the sample.
FIG. 4 shows the results obtained when the plasmid pTIA-1 or its control pCtrl was injected into CHANG cell line of human liver cell line, siTIA-1 or its control siCtrl, which is a siRNA targeting TIA-1, Expression was measured by Western blotting.
FIG. 5 is a graph showing the quantitative analysis of the expression pattern of MFF protein according to an increase in expression of TIA-1 protein in normal liver tissues and liver cancer tissue samples isolated from 9 patients, respectively.
FIG. 6 (a) shows the expression of mitochondrial cytokines when injected with siCI-1 or its control siCtrl, which is an siRNA targeting TIA-1, into a human liver cell line, CHANG cell line, with plasmid pTIA-1 or its control pCtrl, And morphological changes were observed through a fluorescence microscope. (b) shows the number of cells with unchanged mitochondria and the number of cells with long or shortened mitochondria when the cells were sorted according to the morphological changes of the mitochondria in the fluorescence microscopic observation, (%).
Fig. 7 (a) shows the expression of mitochondrial cytokines when injected with the plasmid pTIA-1 or its control pCtrl into a CHANG cell line, which is a human liver cell line, or with siTIA-1 or its control siCtrl, which is a siRNA targeting TIA- The membrane potential difference was measured using a membrane potential difference kit and numerical value was obtained by fluorescence generated at this time. In addition, the results of deprotonating mitochondria by treatment with FCCP (carbonyl cyanide p-trifluoromethoxyphenyl hydrazone) are also shown. (b) is a graph showing the results of measuring the amount of ATP, which is a product of intracellular mitochondria, in each cell line.
Figures 8A-8D show Kaplan-Meier survival curves for different TIA-1 expression in lung cancer patients. The red color represents the TIA-1 high expression group and the green color represents the TIA-1 low expression group. FIG. 8A shows the results of the analysis in the GEO database registration number GSE 5843, FIG. 8B shows the GEO database registration number GSE 11117, FIG. 8C shows the GEO database registration number GSE 14814, and FIG. 8D shows the data set of the GEO database registration number GSE 41271.
FIG. 9A shows the Kaplan-Meier survival curve according to the difference in TIA-1 expression in the lung cancer patient group and shows the analysis result in the data set of the GEO database registration number GSE14814. The red color represents the TIA-1 high expression group and the green color represents the TIA-1 low expression group.
FIG. 9B shows the Kaplan-Meier survival curve according to the difference in MFF expression in lung cancer patients and shows the analysis result in the data set of GEO database registration number GSE14814. The red color represents the MFF high group and the green color represents the MFF low expression group.
FIG. 10A shows the Kaplan-Meier survival curves according to differences in TIA and MFF expression in lung cancer patients and shows the analysis results in the data set of GEO database registration number GSE14814. Black represents TIA-1 low expression and MFF low expression group, blue represents TIA-1 high expression and MFF low expression group, and red color represents TIA-1 high expression and MFF high expression group.
FIG. 10B shows the Kaplan-Meier survival curves according to differences in TIA and MFF expression in lung cancer patients and shows the analysis results in the data set of GEO database registration number GSE14814. Black represents TIA-1 low expression and MFF low expression group, blue represents TIA-1 high expression group (low expression of MFF, TIA-1 high expression group regardless of high expression), red color represents TIA -1 < / RTI > high expression and MFF high expression group.
FIG. 11A shows the Kaplan-Meier survival curve according to the difference of TIA-1 expression in the normal karyotype AML patient group and shows the analysis result in the data set of GEO database registration number GSE12417. The red color represents the TIA-1 high expression group and the green color represents the TIA-1 low expression group.
FIG. 11B shows the Kaplan-Meier survival curve according to the difference in MFF expression in the normal karyotype acute myelogenous leukemia patient group, and shows the analysis result in the data set of GEO database registration number GSE12417. The red color represents the MFF high group and the green color represents the MFF low expression group.
Figure 12a shows the Kaplan-Meier survival curves according to differences in TIA and MFF expression in normal karyotype acute myelogenous leukemia patients and shows the analysis results in the data set of GEO database registration number GSE12417. Black represents TIA-1 low expression and MFF low expression group, blue represents TIA-1 high expression and MFF low expression group, and red color represents TIA-1 high expression and MFF high expression group.
Figure 12B shows the Kaplan-Meier survival curves according to differences in TIA and MFF expression in normal nucleated acute myelogenous leukemia patients and shows the analysis results in the data set of GEO database registration number GSE12417. Black represents TIA-1 low expression and MFF low expression group, blue represents TIA-1 high expression group (low expression of MFF, TIA-1 high expression group regardless of high expression), red color represents TIA -1 < / RTI > high expression and MFF high expression group.
Figure 12c shows the Kaplan-Meier survival curves according to differences in TIA and MFF expression in normal karyotype acute myelogenous leukemia patients and shows the analysis results in the data set of GEO database registration number GSE12417. Black represents TIA-1 low expression and MFF low expression group, blue represents TIA-1 high expression and MFF low expression group, and red color represents TIA-1 high expression and MFF high expression group.
Figure 12d shows the Kaplan-Meier survival curves according to differences in TIA and MFF expression in normal karyotype acute myelogenous leukemia patients and shows the analysis results in the data set of GEO database registration number GSE12417. Black represents TIA-1 low expression and MFF low expression group, blue represents TIA-1 high expression group (low expression of MFF, TIA-1 high expression group regardless of high expression), red color represents TIA -1 < / RTI > high expression and MFF high expression group.
Figure 13a shows Kaplan-meier survival curves according to differences in TIA-1 expression in patients with chronic lymphocytic leukemia and shows the analysis results in the data set of GEO database registration number GSE22762. The red color represents the TIA-1 high expression group and the green color represents the TIA-1 low expression group.
Figure 13b shows Kaplan-meier survival curves according to differences in MFF expression in patients with chronic lymphocytic leukemia and shows the analysis results in the data set of GEO database registration number GSE22762. The red color represents the MFF high group and the green color represents the MFF low expression group.
Figure 14a shows Kaplan-meier survival curves with differences in TIA and MFF expression in patients with chronic lymphocytic leukemia and shows the results of analysis in the data set of GEO database registration number GSE22762. Black represents TIA-1 low expression and MFF low expression group, blue represents TIA-1 high expression and MFF low expression group, and red color represents TIA-1 high expression and MFF high expression group.
FIG. 14B shows the Kaplan-Meier survival curves with differences in TIA and MFF expression in patients with chronic lymphocytic leukemia and shows the results of analysis in the data set of GEO database registration number GSE22762. Black represents TIA-1 low expression and MFF low expression group, blue represents TIA-1 high expression group (low expression of MFF, TIA-1 high expression group regardless of high expression), red color represents TIA -1 < / RTI > high expression and MFF high expression group.

The present invention relates to a composition for estimating cancer diagnosis or prognosis, comprising a substance for measuring the expression level of TIA-1 gene or MFF gene, a kit for cancer diagnosis or prognosis estimation, a cancer diagnosis or prognosis estimation method, A method for detecting a biomarker is provided. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the drawings.

As used herein, the term "diagnosis" is intended to include determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, Determining the prognosis of a stuck subject (e.g., identifying a pre-metastatic or metastatic cancerous condition, determining a stage of a cancer, or determining the response of a cancer to treatment), or determining therametrics And monitoring the status of the object to provide information about the object.

The term "prognosis" refers to predicting various conditions of a patient such as the likelihood of cancer cure after cancer has been diagnosed, the likelihood of recurrence after treatment, and the survival probability of the patient. The prognosis of cancer can be estimated from various perspectives, but it can be judged from the viewpoint of possibility of recurrence, possibility of survival, disease free survival. For the purposes of the present invention, the prognosis may mean the prognosis of survival after diagnosis of cancer. The use of the biomarker provided by the present invention can more easily predict the survival prognosis of a cancer patient and can be used to classify a patient in a high-risk group or determine whether to use a further necessary treatment method, Thereby contributing to the later survival rate.

The term "estimation" relates to the likelihood and / or likelihood that a patient will respond favorably or non-favorably to a therapy and survive after treatment of the patient. The method of the present invention can be used clinically to make treatment decisions by selecting the most appropriate treatment regimen for cancer-causing patients. The estimation method of the present invention can also be used to confirm that the patient preferentially responds to treatment regimens such as, for example, a prescribed treatment or combination, surgical intervention, chemotherapy or the like, Can be used to predict whether or not long-term survival is possible.

In the present invention, the cancer may be exemplified as liver cancer, lung cancer or blood cancer. Blood cancers include leukemia such as acute myelogenous leukemia (e.g., normal karyotype acute myelogenous leukemia) or lymphoid leukemia (e.g., lymphocytic leukemia).

In the present invention, the term "(bio) marker, marker for diagnosis or diagnosis marker" refers to a substance capable of distinguishing cancer cells or tissues from normal cells or tissues. An organic biomolecule such as a polypeptide or a nucleic acid (for example, mRNA or the like) showing an increase pattern in a cell having an antigen, a lipid, a glycolipid, a glycoprotein, a sugar (monosaccharide, a disaccharide, an oligosaccharide and the like) For the purpose of the present invention, the cancer diagnostic marker is a gene in which the expression of TIA-1 gene or a nucleotide (including fragment thereof) of MFF gene or a protein (including fragment thereof) encoded thereby is increased in cancer cells. These markers may be mRNA for any gene or any protein encoded by the gene, or may be a complex marker containing two or more of these markers.

More specifically, the present invention provides TIA-1 and / or MFF as markers for the diagnosis or prognosis estimation of cancer.

The nucleotide sequence of the TIA-1 gene or the amino acid sequence of the protein encoded thereby can be found in a known gene database. For example, the nucleotide sequence of the human TIA-1 gene can be found at NCBI Genbank Accession No. NM_022173.2, and the amino acid sequence of the TIA-1 protein can be found at NCBI Genbank Accession No. NP_071505.2. Preferably, the nucleotide sequence of the TIA-1 gene is set forth in SEQ ID NO: 1, and the amino acid sequence of TIA-1 is set forth in SEQ ID NO:

The nucleotide sequence of the MFF gene or the amino acid sequence of the protein encoded thereby can be found in a known gene database. For example, the nucleotide sequence of the human MFF gene can be found at NCBI Genbank Accession No. NM_001277061.1, and the amino acid sequence of the MFF protein can be found at NCBI Genbank Accession No. NP_001263990.1. Preferably, the nucleotide sequence of the MFF gene is set forth in SEQ ID NO: 3 and the amino acid sequence of MFF is set forth in SEQ ID NO: 4.

However, the above sequence information represents a representative example, and isoforms substantially exhibiting activity equivalent to TIA-1 or MFF can also be included in the scope of the present invention.

As used herein, a "polypeptide" (or protein) is interpreted to include an amino acid sequence that exhibits substantial identity to the amino acid sequence of interest. The above-mentioned substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence as much as possible, and analyzing the aligned sequence using the algorithm commonly used in the art, at least 60% Homology, more preferably at least 80% homology, most preferably at least 90% homology.

For example, the polypeptide may comprise at least about 60%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4% , 99.5% or more, or 99.9% or more identity, and has a biomarker function for the diagnosis or prognosis of liver cancer. In general, the higher the percent identity, the more preferable.

Also, a polypeptide having the above identity includes a polypeptide associated with the beta-adipate pathway, including amino acid sequences in which one or more amino acid residues are deleted, substituted, inserted, and / or added in the polypeptide of the specified amino acid sequence described do. In general, the smaller the number of deletions, substitutions, insertions, and / or additions, the more preferable.

As used herein, the term "polynucleotide" (or nucleotide, nucleic acid) encompasses both DNA (gDNA and cDNA) and RNA molecules. In the nucleic acid molecule, the nucleotide as a basic constituent unit is not only a natural nucleotide, Or analogues in which the base moiety is modified.

The polynucleotide of the present invention is not limited to a nucleic acid molecule encoding the above-described specific amino acid sequence (polypeptide), and may be a polynucleotide having an amino acid sequence exhibiting substantial identity to a specific amino acid sequence as described above or a poly Is interpreted to include nucleic acid molecules encoding the peptides. The above substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence to the greatest correspondence and analyzing the aligned sequence using algorithms commonly used in the art to obtain a homology of at least 60% , More preferably at least 80% homology, and most preferably at least 90% homology.

The polypeptide having the corresponding function includes, for example, a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added. Such a polypeptide comprises a polypeptide involved in the synthesis of 3-hydroxypropionic acid, wherein the polypeptide comprises an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, and / or added, , The number of insertions and / or additions is preferably small. Also, the polypeptide has an amino acid sequence having an identity of about 60% or more with the specific amino acid sequence described above, and includes a polypeptide having a biomarker function for the diagnosis or prognosis of liver cancer. The higher the identity, the more preferable .

The term " complementary "or" complementarity ", as used herein, refers to the ability of purine and pyrimidine nucleotides to bind through hydrogen bonding to form double stranded polynucleotides, including partially complementary. The following base pairs are associated with complementarity: guanine and cytosine; Adenine and thymine; And adenine and uracil. "Complementary" applies substantially to all base pairs in which the above-mentioned relationship includes two single-stranded polynucleotides across the molecule of the full length. "Partially complementary" means that a portion of one of the molecules remains as a single strand because the length of one of the two single-stranded polynucleotides is short.

The composition for cancer diagnosis or prognosis estimation according to the present invention may include a substance for measuring the expression of TIA-1 gene or MFF gene, or a substance for measuring the expression of the two genes, respectively.

The expression measurement or detection in the present invention includes quantitative and / or qualitative analysis, and includes detection of presence and absence and detection of the expression level. Such methods are well known in the art, and those skilled in the art will appreciate You will be able to choose the appropriate method.

In the present invention, the substance for measuring the expression level of the gene may be a substance that detects at least one of the presence, abundance, and presence pattern of the mRNA transcribed by the gene, the protein encoded by the gene, or both .

The present invention may be used to diagnose or prognose cancer by quantitatively and / or qualitatively detecting the gene at a nucleic acid level, particularly at an mRNA level. In the present invention, the substance for measuring the expression level of the gene may be a primer that specifically binds to at least one selected from the group consisting of a nucleotide sequence of the gene, a complementary sequence thereof, a fragment of the nucleotide and a complementary sequence thereof, , An aptamer, and an antisense.

For example, in order to measure the presence and amount or pattern of the mRNA of the gene by RT-PCR, it includes a probe and / or a primer pair specific to the mRNA of the gene. Primer or probe refers to a nucleic acid sequence having a free 3 'hydroxyl group capable of complementarily binding with a template and allowing the reverse transcriptase or DNA polymerase to initiate replication of the template. The gene expression measuring substance used in the present invention may be labeled as a coloring, luminescent or fluorescent substance for signal detection. For example, Northern blot or reverse transcription PCR (polymerase chain reaction) is used for mRNA detection. In the latter case, it is possible to detect a specific gene in a specimen by isolating RNA of the specimen, specifically mRNA, synthesizing cDNA therefrom, and then using a specific primer or a combination of a primer and a probe to detect the presence / Or the amount of expression can be determined.

In the present invention, the substance for measuring the expression level of the gene is a substance specifically expressed in at least one of a polypeptide encoded by the nucleotide sequence, a polypeptide encoded by the complementary sequence, and a polypeptide encoded by a fragment of the nucleotide sequence A peptide fragment, a ligand, a peptide nucleic acid (PNA), an aptamer, an avidity multimer, and a peptidomimetic (Peptidomimetics).

In the present invention, the substance for measuring the expression level of the gene may be any one selected from the group consisting of the presence, presence and presence pattern of the binding body of a protein encoded by the TIA-1 gene or MFF gene, or a messenger RNA (mRNA) Or more.

In the present invention, the substance for measuring the expression level of the gene may include a substance used in various gene (biomarker) detection methods known in the art, for example, a reverse transcription polymerase chain reaction, a competitive polymerase chain reaction (RNase, S1 nuclease assay), in situ hybridization method, DNA microarray method, northern blotting, Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, immunodiffusion , Immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS, mass spectrometry, and protein microarray methods.

That is, the present invention measures the expression of the gene using quantitative / qualitative analysis methods for various nucleic acids / proteins known in the art. For example, RT-PCR / polymerase chain reaction, competitive RT-PCR, real-time RT-PCR, and Nuclease protection assay (NPA) for detection, expression level or pattern detection at the RNA level For example, methods using RNase, S1 nuclease analysis, in situ hybridization, DNA microarray, chip or Northern blot can be used, and these assays are well known and can also be performed using commercially available kits, It is possible to select an appropriate one for the implementation of the present application.

The present invention also provides a kit or system for cancer diagnosis or prognosis estimation comprising the above-described composition for cancer diagnosis or prognosis estimation.

The concrete contents are as described above. The kit according to the present invention may be a variety of kits known in the art, such as a microarray, a gene amplification kit, an immunoassay kit, a luminex assay kit, a protein microarray kit, and an ELISA kit It can be more than one.

The present invention also relates to a method for diagnosing or prognosing a hepatocellular carcinoma, comprising the steps of: Measuring the expression level of the TIA-1 gene or the MFF gene from the biological sample; And comparing the gene expression level measurement result with a reference value.

For example, if the expression level of the TIA-1 gene and / or MFF gene is higher than the reference value, the cancer can be diagnosed as having an onset.

In another example, the expression level of the TIA-1 gene and / or MFF gene may be estimated to be poor (or low) when the level of expression is higher than the reference value, and conversely, the expression level of the TIA-1 gene and / If it is lower than the reference value, it can be estimated that the survival prognosis is good (or high).

A biological sample herein refers to a substance or mixture of substances, including one or more components capable of detecting biomarkers, including but not limited to cells, tissues or body fluids derived from an organism, particularly human, such as whole blood, urine, plasma, and serum But is not limited thereto. As well as cells or tissues cultured in vitro, as well as those directly derived from the organism. A variety of samples may be used to detect cancer biomarkers in accordance with the present application, but are not limited thereto. In one embodiment, urine, whole blood, serum and / or plasma may be used. In other embodiments, cell cultures may be used, including, but not limited to, tissue / cell or invitrocells obtained from, or susceptible to, or suspected of having cancer. It also includes fractions or derivatives of the blood, cells or tissues. When a cell or tissue is used, the cell itself or a fusion of cells or tissues may be used.

The gene expression level in the cancer diagnosis or prognosis estimation method according to the present invention can be determined by various quantitative / qualitative measurement methods for nucleic acids / proteins known in the art. For example, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization method, DNA microarray method, northern blotting, Western blotting, ELISA Enzyme Linked Immuno Sorbent Assay), radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry and protein microarray .

In the cancer diagnosis or prognosis estimation method according to the present invention, the cancer gene can be diagnosed or the prognosis can be estimated by comparing the gene expression level measurement result with the control group measurement result. Or to compare cancer with a predetermined cut-off value to diagnose cancer or to estimate a prognosis. As the control group or the reference group, a sample derived from a normal test sample as a negative control group or a patient-derived sample treated after cancer treatment, a patient-derived sample judged cancerous by a method other than the marker according to the present invention as a positive control group can be used. For example, samples from healthy persons, normal tissue samples from cancer-determining patients, and patients from patients who have received treatment after being diagnosed with cancer may be used as a control or reference group and used for comparison of profiles obtained. For example, the TIA-1 gene or MFF gene, which is an oncological biomarker according to the present invention, is expressed in cancer cells and tissues in comparison with normal cells and tissues, and the expression level of either one or both genes is overexpressed The prognosis is not good.

A variety of methods known in the art can be used to compare the marker profile between the control and sample groups. For example, a digital image comparison of the expression profile and a comparison using the DB for the expression data can be referred to. The profile obtained through marker detection according to the present application can be processed using known data analysis methods. For example, nearest neighbor classifier, partial-least squares, SVM, AdaBoost, and clustering-based classification methods can be used. In order to confirm the significance of the method for estimating the prognosis of liver cancer according to the present invention, various statistical processing methods can be used. As a statistical processing method, a logic regression method can be used in one embodiment. Statistical analysis can also be used to determine the level of confidence regarding the significant differences between the test substance and the control group to diagnose cancer. The raw data used in the statistical processing are the values analyzed by double, triple or multiple for each marker. This statistical analysis method is very useful for making clinically meaningful judgment through statistical treatment of biomarkers as well as clinical and genetic data.

The cancer diagnosis and prognosis estimation method according to the present invention can be used to determine the severity of cancer. For example, mild, moderate or severe, as compared to profiles of positive and negative controls. Furthermore, the marker profile analysis for a certain cancer group can be performed, and classified according to a certain criterion based on the profile result.

The cancer diagnosis and prognosis estimation method according to the present invention can be performed several times over a certain period of time, for example, over a certain period of time, and can be used for monitoring the change in the expression pattern. Depending on the type of marker, an increase or decrease in expression may be associated with the status of the cancer. Can be used to determine HCC incidence, progression, exacerbation, or the like, by comparison with the previous test value or the control group value for the same subject. Based on changes in biomarkers over time, prophylactic measures can be taken to prevent progression of liver cancer. Furthermore, it can be used in conjunction with conventional cancer diagnostic methods such as ultrasound, computerized axial tomography (CT scan) or magnetic resonance imaging (MRI) to confirm cancer.

The present invention relates to a method for detecting a cancer diagnosis or a biomarker for prognosis estimation by measuring the expression level of TIA-1 gene or MFF gene in a biological sample of a human in order to provide information necessary for diagnosis or prognosis estimation of cancer . In the method for detecting the cancer marker for diagnosis or prognosis estimation according to the present invention, the expression level of the gene may be measured by a method for quantitatively / qualitatively measuring various nucleic acids / proteins known in the art. For example, as described above, the reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time PCR, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, DNA microarray, (ELISA), radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS, mass spectrometry, and protein microarray One or more methods can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

1. Experimental preparation

(1) Preparation of sample

The human-derived samples used in this experiment were purchased from Catholic Central Medical Center Catholic Research Institute Sample Bank and used for the experiments. We obtained normal liver and liver cancer tissues from 9 patients and used them for experiments. The experiments were conducted in compliance with the regulations of the Catholic University Institutional Ethics Committee. The human liver cell line, CHANG cell line, was obtained from the ATCC (American Cell Line Bank).

(2) Cell culture

DMEM culture medium containing 10% bovine serum was used for culturing all the cell lines used in the experiment. Cell lines were cultured in an incubator maintained at 37 ° C in an environment maintained at 5% carbon dioxide concentration. The intracellular injection of the outer genome was performed using a 35 mm ² culture dish, and the mitochondrial function assay was performed using a 96-well culture dish. In all other situations, cell lines were cultured and maintained in 100 mm ² culture dishes.

2. Experimental Method

(1) Analysis of gene expression changes in cells and tissues

Invitrogen trizol solution was used for analysis of gene expression and changes in normal liver and liver cancer tissues. After the tissue crushing and cell collection, the triose solution was treated to obtain mRNA, and cDNA was synthesized using Toyobo's cDNA synthesis kit. Real-time quantitative PCR method was used to quantify the gene expression and changes in the experimental and control groups.

(2) Analysis of protein expression in cells

From the collected cells, proteins in the cells were obtained using RIPA (radio-immunoprecipitation assay) solution, and the same total amount of proteins was separated by size in electrophoresis in SDS-PAGE gel. The specific size of the separated proteins was transferred to the PVDF membrane, and the antibody of the protein was attached to the PVDF membrane. After the secondary antibody, which can quantify the expression level of a specific protein, was treated, the light irradiated from the specific protein was recorded on the X-ray film to quantitatively analyze the change of the protein to be observed.

(3) intracellular infusion of external genome transporters

A total of two plasmid-shaped outer genomes and two siRNA-type outer genomes were used for the experiment through intracellular injection. Two siRNAs were purchased from Genolution (siCtrl, siTIA-1), and two plasmids were prepared by inserting TIA-1 into the back of the HA protein based on the pcDNA3-HA transporter (referred to as pTIA-1), and the pcDNA3-HA transporter (pCtrl) was used as a control for the plasmid-type outer genome.

The injection of all external genomes used in the experiments was carried out using Lipofectamine 2000 from Invitrogen. Each outer dielectric was treated with Lipofectamine 2000 as a manufacturer's manual according to the experiment and then mixed with the cell culture solution to inject the outer genome into the cells.

(4) Mitochondrial morphological analysis

In order to observe the morphological changes of the mitochondria, two experiments were conducted. First, in order to observe mitochondrial-specific morphological changes, Mitochondria were stained with Invitrogen mitotracker, and then a fluorescence microscope from Carl Zeiss was used. The mitotracker reagent was mixed with the cell culture medium for 30 minutes according to the manual of the manufacturer, and morphological changes were observed through fluorescence expressed specifically in mitochondria. Electron microscopy was used to confirm direct morphological changes of the mitochondria. In the case of electron microscopy, the cells were immobilized with 1% osmium tetroxide, embedded in Epon 812, and subjected to ultrathin sections using an electron microscope of JEM 1010 Respectively.

(5) Mitochondrial function analysis

In order to measure the functional changes of mitochondria, two experiments were conducted. First, the mitochondrial membrane potential was measured to confirm the direct mitochondrial function change, and the amount of ATP, the product of mitochondria, was determined. For the measurement of the mitochondrial membrane potential difference, the control and experimental cell lines were cultured in the same number of wells in a 96-well culture dish, and then measured using a kit for measuring the mitochondrial membrane potential difference using Abcam. JC-1 was mixed with the cell culture solution for 10 minutes according to the manufacturer's manual, and the fluorescence measured at a specific wavelength of 535 nm (excitation) / 590 nm (emission) was quantified. When measuring the amount of intracellular ATP, the cell lines of the control and experimental groups were cultured in the same number of wells in a 96-well culture dish using Promega's Mitochondrial ToxGlo assay kit, and then exposed to a culture medium containing galactose for 90 minutes according to the manufacturer's manual, The amount of ATP in the cells was determined by measuring the degree of luminescence with the reagent.

(6) Analysis of gene expression changes in human liver cancer stage

We analyzed gene expression and change pattern of human liver cancer tissues classified by lesion according to TNM (Tumor-Node-Metastasis) classification using RNA sequencing method. Liver cancer stage was classified as TG1 (Tumor grade 1), TG2 (Tumor grade 2), and TG3 (Tumor grade 3) depending on the degree of tumor differentiation. The degree of tumor differentiation indicates morphologically or functionally the degree to which the shape and function of tumor cells resemble normal cells, and that the degree of depletion of cells deviating from normal cells increases from grade 1 to grade 3 (ie, ), The lower the degree of differentiation, the higher the degree of malignancy and the course is poor after treatment. The gene expression and change patterns were analyzed statistically by the numerical values of the gene expression levels measured in the data.

(7) Survival analysis by difference of expression of TIA-1 and MFF in various cancers

The survival rates of TIA-1 and / or MFF and TIA-1 and / or MFF expression were compared by computer program to confirm the relationship between the degree of TIA-1 and / or MFF expression and survival prognosis. The survival and statistical analysis were performed using the algorithm provided by the web - based PROGgeneV2 program. Information on each cancer patient group is available for GEO (Gene Expression Omnibus) registration numbers GSE5843, GSE11117, GSE41271 and GSE14814 for lung cancer patients, GSE12417 for normal karyotype AML patients, and Chronic lymphocytic leukemia) were used for the data set of GSE22762.

3. Experimental Results

(1) an increase in the expression of TIA-1 mRNA in cancer

In order to confirm the expression pattern of TIA-1 mRNA in cancer, the expression of TIA-1 mRNA using human normal liver tissue (HCC) and hepatocellular carcinoma (HCC) was confirmed by RNA sequencing Respectively. As a result, as shown in FIG. 1 (a), the expression of TIA-1 mRNA was significantly increased in liver cancer tissues compared with normal liver tissues. The expression of TIA-1 mRNA was more increased in liver cancer stage . In addition, as shown in Fig. 1 (b), it was confirmed that TIA-1 mRNA expression was significantly increased in liver cancer tissues compared with normal liver tissues. Thus, the expression of TIA-1 mRNA is increased in cancer progression.

(2) confirmation of increased expression of TIA-1 protein in cancer

In order to confirm the expression pattern of TIA-1 protein in cancer development, the expression of TIA-1 protein in human normal liver and liver cancer tissues was measured by Western blotting. As a result, as shown in FIG. 2, it was confirmed that expression of TIA-1 protein was increased in liver cancer tissues compared with normal tissues in all the samples isolated from nine patients.

(3) confirmation of increased expression of MFF protein in cancer

In order to confirm the expression pattern of MFF protein in cancer development, expression of MFF protein was measured by Western blot using human normal liver tissue and liver cancer tissue. As a result, as shown in FIG. 3, it was confirmed that expression of MFF protein was increased in liver cancer tissues compared with normal tissues in all the samples isolated from 9 patients.

(4) Confirmation of increase of MFF protein expression by TIA-1

In order to confirm that the increase of MFF protein expression is the result of the regulation of TIA-1, the expression level of TIA-1 was changed in CHANG cell line of human liver cell line, and the degree of MFF expression in the same experimental group and control group was confirmed. Specifically, the expression of TIA-1 was increased by injecting plasmid pTIA-1 into a human liver cell line, or the siRNA-1 siRNA targeting TIA-1 was injected to reduce the expression of TIA-1 PCtrl or siCtrl was injected as a control group. As a result, as shown in FIG. 4, when the expression of TIA-1 was increased, the expression of MFF was increased, and when the expression of TIA-1 was decreased, the expression of MFF was also decreased. Thus, it was found that the increase of MFF protein expression was the result of the regulation of TIA-1.

Next, in order to examine the effect of expression of TIA-1 protein on the expression of MFF protein when cancer was generated in the body, expression pattern of MFF protein according to increase of TIA-1 protein expression was quantified and analyzed. As a result, as shown in FIG. 5, it was confirmed that the MFF protein was increased in a similar manner as the expression of TIA-1 protein increased in liver cancer tissues compared to normal liver tissues (FIG. 5).

(5) Confirmation of morphological changes of mitochondria by TIA-1

In order to confirm that the expression of TIA-1 induces a morphological change in mitochondria closely related to cell activity, morphological changes of mitochondria were examined by fluorescence microscopy after controlling the expression of TIA-1 protein in human liver cell line CHANG cell line Respectively. Specifically, the expression of TIA-1 was increased by injecting plasmid pTIA-1 into a human liver cell line, or the siRNA-1 siRNA targeting TIA-1 was injected to reduce the expression of TIA-1 PCtrl or siCtrl was injected as a control group. As a result, as shown in FIG. 6, when the expression of TIA-1 is increased, the number of cells showing a morphologically shortened length of mitochondria increases, and when the expression of TIA-1 is suppressed, the length of mitochondria becomes longer And it was confirmed that morphologically changed cells were increased.

(6) Identification of functional changes of mitochondria by TIA-1

In order to confirm that the expression of TIA-1 regulates the functional changes of mitochondria closely related to cell metabolism, the expression of TIA-1 protein in human liver cell line CHANG cell line is regulated, and mitochondrial membrane associated with mitochondrial function Potential difference and intracellular ATP amount were measured. Specifically, the expression of TIA-1 was increased by injecting plasmid pTIA-1 into a human liver cell line, or the siRNA-1 siRNA targeting TIA-1 was injected to reduce the expression of TIA-1 PCtrl or siCtrl was injected as a control group. As a result, as shown in FIG. 7, when the expression of TIA-1 was increased, the mitochondrial membrane potential difference and intracellular ATP amount were significantly decreased compared with the control group, whereas when the expression of TIA-1 was decreased, Potential difference and intracellular ATP levels were significantly increased compared to the control group. Thus, it was confirmed that the expression of TIA-1 modulates the function of mitochondria.

(7) Increased survival rate according to difference of TIA-1 and MFF expression in various cancers

The survival rates of TIA-1 and / or MFF and TIA-1 and / or MFF expression were compared by computer program to confirm the relationship between the degree of TIA-1 and / or MFF expression and survival prognosis.

Survival analysis of patients with lung cancer showed that the overall survival (OS) was lower as the expression of TIA-1 was increased in lung cancer patients of various cohorts, while the expression of TIA-1 was decreased It was confirmed that the survival rate of the patient was further increased (see Figs. 8a, 8b, 8c and 8d). In addition, it was confirmed that as the expression of TIA-1 increases, the survival rate decreases as the expression of MFF increases in the experimental group in which the survival rate is lowered. That is, the lower the expression of TIA-1 and / or MFF, the better the survival prognosis and the higher the expression of TIA-1 and / or MFF, the worse the survival prognosis was (see Figs. 9a, 9b, 10a and 10b) ).

Survival analysis of patients with normal karyotype AML showed that overall survival (OS) decreased as TIA-1 expression increased and survival rate decreased as TIA-1 expression increased The survival rate was decreased as the expression of MFF increased. 11a, 11b, 12a, 12b, and 12b show that the survival prognosis is better as the expression of TIA-1 and / or MFF is lower and the survival prognosis is worse as the expression of TIA-1 and / 12c and 12d).

In addition, the survival analysis of patients with chronic lymphocytic leukemia showed that overall survival (OS) decreased as TIA-1 expression increased, and that MFFs decreased as TIA-1 expression increased And the survival rate was lowered as the expression of the gene was increased. That is, the lower the expression of TIA-1 and / or MFF, the better the survival prognosis, and the higher the expression of TIA-1 and / or MFF, the worse the survival prognosis was (see FIGS. 13A, 13B, 14A and 14B ).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry Academic Cooperation Foundation of Catholic University <120> Biomarker for diagnosing or prognosing cancer and uses thereof <130> pn1504-087 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 4653 <212> DNA <213> TIA-1 gene sequence <400> 1 gctcctaggc tcccggctcg ccgccatctt gtattggggt ttcattgttc ccgctgggcc 60 gggcggttta gtgtaattgc cgccggagga ggaggcggag taacctctgg tcagccgaga 120 aaccccacta tcctgtagcc ataaccgctt aaacgatttg ggaggtagtg aagggcaggg 180 agctggacct ggaggcgccg ccgcgacagc agcagccatg gaggacgaga tgcccaagac 240 tctatacgtc ggtaaccttt ccagagatgt gacagaagct ctaattctgc aactctttag 300 ccagattgga ccttgtaaaa actgcaaaat gattatggat acagctggaa atgatcccta 360 ttgttttgtg gagtttcatg agcatcgtca tgcagctgca gcattagctg ctatgaatgg 420 acggaagata atgggtaagg aagtcaaagt gaattgggca acaaccccta gcagtcaaaa 480 gaaagataca agcagtagta ccgttgtcag cacacagcgt tcacaagatc atttccatgt 540 ctttgttggt gatctcagcc cagaaattac aactgaagat ataaaagctg cttttgcacc 600 atttggaaga atatcagatg cccgagtggt aaaagacatg gcaacaggaa agtctaaggg 660 atatggcttt gtctcctttt tcaacaaatg ggatgctgaa aacgccattc aacagatggg 720 tggccagtgg cttggtggaa gacaaatcag aactaactgg gcaacccgaa agcctcccgc 780 tccaaagagt acatatgagt caaataccaa acagctatca tatgatgagg ttgtaaatca 840 gtctagtcca agcaactgta ctgtatactg tggaggtgtt acttctgggc taacagaaca 900 actaatgcgt cagacttttt caccatttgg acaaataatg gaaattcgag tctttccaga 960 taaaggatat tcatttgttc ggttcaattc ccatgaaagt gcagcacatg caattgtttc 1020 tgttaatggt actaccattg aaggtcatgt tgtgaaatgc tattggggca aagaaactct 1080 tgatatgata aatcccgtgc aacagcagaa tcaaattgga tatccccaac cttatggcca 1140 gtggggccag tggtatggaa atgcacaaca aattggccag tatatgccta atggttggca 1200 agttcctgca tatggaatgt atggccaggc atggaaccag caaggattta atcagacaca 1260 gtcttctgca ccatggatgg gaccaaatta tggagtgcaa ccgcctcaag ggcaaaatgg 1320 cagcatgttg cccaatcagc cttctgggta tcgagtggca gggtatgaaa cccagtgaat 1380 aaggactcca gaatctaaag ccagtggctt gaggctacag ggagtgtagt aaagccgttg 1440 tttacttaaa gatttatcaa atcagtcagt gcaaatgtca gatacaatgt atttatttaa 1500 aagattcatt tttaatcatg aaattactta tcatccacat tgttttaaaa agaaacaaga 1560 tgctggatgt ctgccaattt ttgccttcat tacctttttt gataaagttt ctcagatcct 1620 tgtttcaaac acaaatgcag ggattgctgc cactttttaa ctattaagag gcagaaaatt 1680 gcacaatatt gaactttttt ccactgaagt agtgtgcagt tctagtttgc attcctgata 1740 tgatttaaaa catgtaatat aaagatgtta aaaaaaaaaa ccaaaactgt gcagagtcta 1800 gaagttgttt gtcatcttca gcttgtgcac aattctgttt taggttaaaa aaaggcattg 1860 tttgagctgt cccatctcca ctgttatccc tttggggttt tttaatataa attattagtt 1920 tacatcattt ttgtatctac atcttttttc acaaatttgt cttgccttat taaagttctg 1980 taaaatatac ttaaatggaa aaaatgatgt tcatttagat tgaaaacttt tctcagatgg 2040 attgataatt gcattcatct tgtgttttat atgagaaggt gcctcaagaa tttcctgttg 2100 gatttgttta aaaggatttt tatctttcgt gataaacttt gctgtgtacc aggaactata 2160 aaaacaaaaa cttgttacta aagaaaatat ctgaaatgtg ataagttctt atgccatgtt 2220 aatttcatgt gtcaacttca acatttacat gtattatttc attatgtaaa atgttttagc 2280 aatttaatat tttgcacagt tagcaaactt tgtatgtcat ttccttcaag gcatcatgca 2340 gagttgacat gagatttata aggttttaag ttgtttgcat gtgaaaatca aatacatact 2400 ttggtagtct ttgaatacaa agtcatctgc tcttgttttt caagaatttt gagacacaaa 2460 gttgtatgta aaggaatata ttaatttgcc gttttctagg tagatttgct caaaaagagt 2520 gaatcaactt aatatgtaca aatgatagct gtgaaactgt agaatatctt tgtgtcaggc 2580 ttggagttca ttgtgacctc caaattttgc ctgaaggacc agctgggcaa agcatctttt 2640 aaatgttcag aggccaaaag ataaacaaaa aaaaaacctt aaaatcctac ctctttaaac 2700 agccttcaga taagagaatc ctcagtgcaa tcattatttt gattcgtttg gtacctgttt 2760 tcctggagtt cccgatttta ttattttggg gtggctccaa gcattaagag gtttaatctt 2820 tgatggcatt gttctagttt tgaaatttct agtatatttc agagtctctt agaagacttg 2880 tgtgggaagt ttcactttgt tttcagtgaa gatcacaaac ctccttcttc ctttactcaa 2940 gaggaaaggt cccagtatac atatttgaat ggttgatggt tttcaagacc ttcagggagc 3000 tccctgcatt ttacctagaa acagaaaagg cccgcaaaat cttaagtttc ctggcctgca 3060 tttcccgggt aggggcaaat gactccaagc tggtctctaa gccaataccc ttataaacca 3120 gagcccagga aagacagctc gagtgtataa ttctctggag ctcaattcta tgcagttgtg 3180 ctgatatttc attaagtcac tgtgtatttt taagtgttga tacattaaaa gtcgctttat 3240 ggaagatgag taaatttttt aaatacttgg aaattttatt tccttgttaa cttctacaga 3300 tcagggcatg caaccaaaag cagcttaaat gaaattatttt aaaataaaat atcaggaagc 3360 tatttttaga tttcttctgg cttatgtttc tactttagga ccctcattgt tctcttatta 3420 aaaaaaatta tttcctgtgc atctcatgga ctgcagggta aattatttgg gcataaataa 3480 tttaaatagt tttctttcat tttgactatc tccagtaata acagttttta ttatccagca 3540 tattggctta ttgcacaaat cttaaaatgt acattgacta ctttctgaga agaaagtggt 3600 atcagtactc atgatgaaaa ggttactact gaacaaattc acatttcagg aacacctcta 3660 tttttggttt aaatcttact cttagttttt ccgtctaaaa atcatactgg tattagtatc 3720 aggtaaggaa attaaagttt ttaaaatggt ttcattctct gcaatatgca aaatttagat 3780 tttactttct ggtactgtaa agaacctgaa gtgatttaca cttaatgggt gattaatcca 3840 gtattcttta ccctgaatgt ttggatatta aagttccttt atgttttcta taacctgtgg 3900 gatcttcttg cagtgattat tgtgtgtgag attttttttc tttttggtct atccatattg 3960 ttatattcac tcaggtattt tttttttaat cttattccag aatcagtggt ttatattggg 4020 ttactgttta acaccaaatg gaattggcat tctgcagatt taattaatta tgaaaccagg 4080 gtctcatttt ccttgctgat acttgttgaa aatgagattc acattctagt ctttattttc 4140 ctcctgtttt gtccctgtgc ttgtacatct tccttttatt tgtgtgttat agttctattc 4200 catttgagaa ggcagttggt aagaactaga ttgcatgtac aaagacaggt ttactaagtg 4260 ctgtacagtg gtcctgaggt tacagttgaa ttagaaaaac gaaatgtact tacaggaaat 4320 aagaaagcaa acctttcaaa tgagagtgat gatttcttta aaaaaaatca gtttttttct 4380 ctcaaataat gttctttatt tcacgaaatc gtcaatctta agcatgagca gggataaaca 4440 actcctagaa ggaactcaat tcattcttcc tggattttct ctgttgttaa atcacaaaaa 4500 tgatagtccc caatcgtttc tttataggag gttattacat ttcattacag tcactgcatt 4560 ttgactgttg tgtttagaat ttgaatgtac atccaaaatg atgagtttca atttaagagc 4620 cttaataaaa tgtgtgagtg tgtctcaatt gaa 4653 <210> 2 <211> 386 <212> PRT <213> TIA-1 amino acid sequence <400> 2 Met Glu Asp Glu Met Pro Lys Thr Leu Tyr Val Gly Asn Leu Ser Arg   1 5 10 15 Asp Val Thr Glu Ala Leu Ile Leu Gln Leu Phe Ser Gln Ile Gly Pro              20 25 30 Cys Lys Asn Cys Lys Met Ile Met Asp Thr Ala Gly Asn Asp Pro Tyr          35 40 45 Cys Phe Val Glu Phe His Glu His Arg His Ala Ala Ala Ala Lea Ala      50 55 60 Ala Met Asn Gly Arg Lys Ile Met Gly Lys Glu Val Lys Val Asn Trp  65 70 75 80 Ala Thr Thr Ser Ser Gln Lys Lys Asp Thr Ser Ser Thr Val                  85 90 95 Val Ser Thr Gln Arg Ser Gln Asp His Phe His Val Phe Val Gly Asp             100 105 110 Leu Ser Pro Glu Ile Thr Thr Glu Asp Ile Lys Ala Ala Phe Ala Pro         115 120 125 Phe Gly Arg Ile Ser Asp Ala Arg Val Val Lys Asp Met Ala Thr Gly     130 135 140 Lys Ser Lys Gly Tyr Gly Phe Val Ser Phe Phe Asn Lys Trp Asp Ala 145 150 155 160 Glu Asn Ala Ile Gln Gln Met Gly Gly Gln Trp Leu Gly Gly Arg Gln                 165 170 175 Ile Arg Thr Asn Trp Ala Thr Arg Lys Pro Pro Ala Pro Lys Ser Thr             180 185 190 Tyr Glu Ser Asn Thr Lys Gln Leu Ser Tyr Asp Glu Val Val Asn Gln         195 200 205 Ser Ser Pro Ser Asn Cys Thr Val Tyr Cys Gly Gly Val Thr Ser Gly     210 215 220 Leu Thr Glu Gln Leu Met Arg Gln Thr Phe Ser Pro Phe Gly Gln Ile 225 230 235 240 Met Glu Ile Arg Val Phe Pro Asp Lys Gly Tyr Ser Phe Val Arg Phe                 245 250 255 Asn Ser His Glu Ser Ala Ala His Ala Ile Val Ser Val Asn Gly Thr             260 265 270 Thr Ile Glu Gly His Val Val Lys Cys Tyr Trp Gly Lys Glu Thr Leu         275 280 285 Asp Met Ile Asn Pro Val Gln Gln Gln Asn Gln Ile Gly Tyr Pro Gln     290 295 300 Pro Tyr Gly Gln Trp Gly Gln Trp Tyr Gly Asn Ala Gln Gln Ile Gly 305 310 315 320 Gln Tyr Met Pro Asn Gly Trp Gln Val Pro Ala Tyr Gly Met Tyr Gly                 325 330 335 Gln Ala Trp Asn Gln Gln Gly Phe Asn Gln Thr Gln Ser Ser Ala Pro             340 345 350 Trp Met Gly Pro Asn Tyr Gly Val Gln Pro Pro Gln Gly Gln Asn Gly         355 360 365 Ser Met Leu Pro Asn Gln Pro Ser Gly Tyr Arg Val Ala Gly Tyr Glu     370 375 380 Thr Gln 385 <210> 3 <211> 2277 <212> DNA <213> MFF gene sequence <400> 3 gtctgcgcat cgcttaccgt ggagccgttc cagtgcgtgg gccacgcgtc cgcccttttc 60 cgcgtcacat gaccacgaac actcttccgc tacggctccc agaaggggcc agcccgcgcc 120 tttcgcgctt ctgccctggc cctctgcggg ccgctccgcc ggtgctgtcc ctgggcgcct 180 ccgtgctctc agccaaccgc ctctgagagc gcccactcga gcgccccggg agccagaggg 240 cgggggtcct cgccgggacc ctcctgtggg cccaggggga caaaagtggc tctcaatcca 300 gcacatgcac attgaagcaa gttaaaggat ttaatatgaa gcacagaagc agatagtgcc 360 aaatagcaag cagtagttgt tacacatttc tggaaaagca gtgtccgtgt tgttatgtac 420 acctccaaaa aaagttcaaa tgtgtagggg aattgttgtg taaagtaaac tgaactgcag 480 ggtagcagta ttttaatagc tattatagac ctgtatttac tgtatttaaa tgagtaaagg 540 aacaagcagt gacacatcac taggaagggt gagcagggca gcatttcctt ctcccactgc 600 tgctgagatg gcagaaatta gtcgaattca gtacgaaatg gaatatactg aaggcattag 660 tcagcgaatg agggtcccag aaaagttaaa agtagcaccg ccaaacgctg acctggaaca 720 aggattccaa gaaggagttc caaatgctag tgtgataatg caagttccgg agaggattgt 780 tgtagcagga aataatgaag atgtttcatt ttcaagacca gcagatcttg accttattca 840 gtcaactccc tttaaacccc tggcactgaa aacaccacct cgtgtactta cgctgagtga 900 aagaccacta gattttctgg atttagaaag acctcctaca acccctcaaa atgaagaaat 960 ccgagcagtt ggcagactaa aaagagagcg gtctatgagt gaaaatgctg ttcgccaaaa 1020 tggacagctg gtcagaaatg attctctgtg gcacagatca gattctgccc caagaaataa 1080 aatttcaagg ttccaggcac cgatttctgc accggagtac actgtgacac catcgccaca 1140 acaggctcgg gtctgtcctc cccatatgtt acctgaagat ggagctaatc tttcctctgc 1200 tcgtggcatt ttgtcgctta tccagtcttc tactcgtagg gcataccagc agatcttgga 1260 tgtgctggat gaaaatcgca gacctgtgtt gcgtggtggg tctgctgccg ccacttctaa 1320 tcctcatcat gacaacgtca ggtatggcat ttcaaatata gatacaacca ttgaaggaac 1380 gtcagatgac ctgactgttg tagatgcagc ttcactaaga cgacagataa tcaaactaaa 1440 tagacgtcta caacttctgg aagaggagaa caaagaacgt gctaaaagag aaatggtcat 1500 gtattcaatt actgtagctt tctggctgct taatagctgg ctctggtttc gccgctagag 1560 gtaacatcag ccctcaaaaa tactgtctca acagctggaa atataaaaga tttgcaaact 1620 tctttgtttc tgtctctgca ttgtatgcca ttttatagtc cacaccctga aaatgtattt 1680 cttccagaaa gtctggagga aggacctata tttgtagaag taaaggtata ttctgtcact 1740 cagctgtatt cacgtctgag cagttctgca gtaacacctg cttaaaattc tccctttgca 1800 tgttttgtaa ataggctcca gttttgtttt ttaaaaggaa tttatttttt gcctcatcag 1860 tccacccaac tgattctgaa tgggagagag tctgtagaga attgattcag aaaagtgtct 1920 gtaaagaaa aacaattatt ttgtcctgtt tctcaaacag tgttaagcag ttttgttaat 1980 agacattttt gcatcgacac ttcaacatta acactttcaa agtcatggtc tggtgccaga 2040 tttaagaaac tcgaaccacc taatatttca taaccttctt cattaggtac ttgtacagat 2100 taatttctaa cattgcagca gtttcatatg tgtgcaatat gtgcattctt tcattttagt 2160 tttgcacttg gttttctata aagtacgttt ttactcagtt catgcgtgaa caatttaaaa 2220 aacgacagaa taaggtacaa atgtagtgta tttaataaac tgtcaaccaa agaagta 2277 <210> 4 <211> 342 <212> PRT <213> MFF amino acid sequence <400> 4 Met Ser Lys Gly Thr Ser Ser Asp Thr Ser Leu Gly Arg Val Val Ser   1 5 10 15 Ala Ala Phe Pro Ser Thr Ala Ala Glu Met Ala Glu Ile Ser Arg              20 25 30 Ile Gln Tyr Glu Met Glu Tyr Thr Glu Gly Ile Ser Gln Arg Met Arg          35 40 45 Val Pro Glu Lys Leu Lys Val Ala Pro Pro Asn Ala Asp Leu Glu Gln      50 55 60 Gly Phe Gln Glu Gly Val Pro Asn Ala Ser Val Ile Met Gln Val Pro  65 70 75 80 Glu Arg Ile Val Val Ala Gly Asn Asn Glu Asp Val Ser Phe Ser Arg                  85 90 95 Pro Ala Asp Leu Asp Leu Ile Gln Ser Thr Pro Phe Lys Pro Leu Ala             100 105 110 Leu Lys Thr Pro Pro Arg Val Leu Thr Leu Ser Glu Arg Pro Leu Asp         115 120 125 Phe Leu Asp Leu Glu Arg Pro Pro Thr Thr Pro Gln Asn Glu Glu Ile     130 135 140 Arg Ala Val Gly Arg Leu Lys Arg Glu Arg Ser Ser Met Ser Glu Asn Ala 145 150 155 160 Val Arg Gln Asn Gly Gln Leu Val Arg Asn Asp Ser Leu Trp His Arg                 165 170 175 Ser Asp Ser Ala Pro Arg Asn Lys Ile Ser Arg Phe Gln Ala Pro Ile             180 185 190 Ser Ala Pro Glu Tyr Thr Val Thr Pro Ser Pro Gln Gln Ala Arg Val         195 200 205 Cys Pro Pro His Met Leu Pro Glu Asp Gly Ala Asn Leu Ser Ser Ala     210 215 220 Arg Gly Ile Leu Ser Leu Ile Gln Ser Ser Thr Arg Arg Ala Tyr Gln 225 230 235 240 Gln Ile Leu Asp Val Leu Asp Glu Asn Arg Arg Pro Val Leu Arg Gly                 245 250 255 Gly Ser Ala Ala Ala Thr Ser Asn Pro His His Asp Asn Val Arg Tyr             260 265 270 Gly Ile Ser Asn Ile Asp Thr Thr Ile Glu Gly Thr Ser Asp Asp Leu         275 280 285 Thr Val Val Asp Ala Ser Leu Arg Arg Gln Ile Ile Lys Leu Asn     290 295 300 Arg Arg Leu Gln Leu Leu Glu Glu Glu Asn Lys Glu Arg Ala Lys Arg 305 310 315 320 Glu Met Val Met Tyr Ser Ile Thr Val Ala Phe Trp Leu Leu Asn Ser                 325 330 335 Trp Leu Trp Phe Arg Arg             340

Claims (10)

A composition for estimating the diagnosis or prognosis of liver cancer or lung cancer, which comprises a substance that measures the expression level of TIA-1 (T-cell restricted intracellular antigen 1) gene and MFF (mitochondrial fission factor) gene. The method according to claim 1,
Wherein the substance for measuring the expression level of the gene measures the amount of the mRNA transcribed by the gene or the protein encoded by the gene. The method according to claim 1,
The expression level of the gene may be measured by a method selected from the group consisting of reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, DNA microarray, Choose between Western blotting, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation, FACS, mass spectrometry and protein microarray methods. Lt; RTI ID = 0.0 &gt; of: &lt; / RTI &gt; delete A kit for estimating the diagnosis or prognosis of liver cancer or lung cancer comprising the composition according to any one of claims 1 to 3. 6. The method of claim 5,
Wherein the kit is at least one selected from the group consisting of a microarray, a gene amplification kit, an immunoassay kit, a luminex assay kit, a protein microarray kit, and an ELISA kit. Treating the composition according to claim 1 to a biological sample;
Measuring an expression level of the TIA-1 gene and the MFF gene from the biological sample; And
And comparing the gene expression level with a reference value. The method for providing information for diagnosis or prognosis estimation of liver cancer or lung cancer. 8. The method of claim 7,
The expression level of the gene may be measured by a method selected from the group consisting of reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, DNA microarray, (ELISA), radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS, mass spectrometry, and protein microarray RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; A method for detecting a biomarker for diagnosis or prognosis estimation of liver cancer or lung cancer, comprising the step of measuring the expression level of TIA-1 gene and MFF gene present in a biological sample. 10. The method of claim 9,
The expression level of the gene may be measured by a method selected from the group consisting of reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, DNA microarray, (ELISA), radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS, mass spectrometry, and protein microarray RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;

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