KR20200109583A - Biomarker composition comprising TXNDC7 for diagnosis or predicting prognosis of hepatocellular carcinoma - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing or predicting a prognosis of hepatocellular carcinoma and including TXNDC7. As a result of analyzing a change in the expression of TXNDC families in hepatocellular carcinoma and normal liver tissue, it has been confirmed that the expressions of mRNA and protein of TXNDC7 increase and the expression of TXNDC11 decreases in tissue with hepatocellular carcinoma compared to in normal liver tissue. In addition, it has been confirmed that an increase in the expression of TXNDC7 and a decrease in the expression of TXNDC11 in hepatocellular carcinoma are related to a poor prognosis of hepatocellular carcinoma. Accordingly, TXNDC7 or TXNDC11 of the present invention is usable as a biomarker for hepatocellular carcinoma, and a diagnosis and a prognosis prediction of hepatocellular carcinoma are possible through an analysis of the expression amount of TXNDC7 or TXNDC11 in Hepatocellular carcinoma. Also, TXNDC7 or TXNDC11 is expected to be useful to develop an agent for preventing or treating hepatocellular carcinoma.

Description

Biomarker composition comprising TXNDC7 for diagnosis or predicting prognosis of hepatocellular carcinoma}

The present invention relates to a biomarker composition for diagnosing hepatocellular carcinoma or predicting prognosis comprising TXNDC7 (thioredoxin-domain-containing protein 7).

Hepatocellular carcinoma (HCC) is the most common form of liver cancer. It is a major cancer that ranks third in cancer incidence in Korea, and has a very poor prognosis with a 5-year survival rate of 9.6%. The incidence of hepatocellular carcinoma increases rapidly from the 40s and peaks in the 60s. However, hepatocellular carcinoma does not have many characteristic symptoms, and this period is a time when social activities are actively carried out, and it may appear early because it is bound to be accompanied by some non-specific symptoms related to cancer such as fatigue, loss of appetite, and general weakness. Symptoms that are present are overlooked, making early detection difficult.

Despite the recent development of diagnostic techniques and treatment methods for hepatocellular carcinoma, effective treatment is difficult because many patients are found to be staged. Therefore, early diagnosis of HCC and performing a curative procedure is the most effective method to increase the patient's survival rate.

The liver is a major organ that has the function of detoxifying various substances that produce reactive oxygen species (ROS), and when the liver's reduction-oxidation (redox) system is disrupted, it causes harmful processes related to oxidative stress and liver cancer. Results. The evidence accumulated so far shows that the initiation and progression of tumors is related to oxidative stress, and in particular, oxidative stress in the liver can promote or worsen tumor formation.

Oxidative stress in the liver promotes hepatocellular carcinoma, and paradoxically, hepatocellular carcinoma induces oxidation-reduction capacity. Induction of the oxidation-reduction ability of hepatocellular carcinoma is to protect itself by regulating the metabolism of reactive oxygen species through various signaling pathways related to tumor formation because cancer cells can be damaged by high reactive oxygen species.

The thioredoxin (TXN) system is indispensable for maintaining the coordination and regulation of redox states in cells. TXNDC (thioredoxin (TXN)-domain containing protein) is a regulator that regulates the redox state and belongs to a group of redox proteins that promote reversible oxidation of cysteine thiol to disulfide. TXNDC is involved in various functions such as redox homeostasis, oxidative stress, disulfide-containing proteins and transcriptional regulation. The TXNDC family is made up of 17 and regulates redox states at distinct intracellular locations including the plasma membrane, cytoplasm, endoplasmic reticulum and nucleus.

However, few studies have investigated the role of TXNDC in cancer progression, especially hepatocellular carcinoma. In previous studies, upregulation of TXNDC5, which is involved in the regulation of protein folding and chaperone activity in cervical cancer, colon cancer, gastric cancer, liver cancer and lung cancer, has been reported (Horna-Terron, E., et al. ., 2014). In addition, overexpression of TXNDC9, which is involved in chaperone-assisted protein folding, is associated with poor prognosis in colorectal cancer (Lu, A., et al., 2012), and is involved in tumor necrosis factor signaling metastasis. It has been reported that TXNDC17 promotes resistance to chemotherapy in ovarian cancer (Zhang, SF, et al., 2015).

Accordingly, the present inventors confirmed that in the process of studying the expression and function of the TXNDC family in HCC, TXNDC7 increased mRNA and protein expression in HCC compared to normal liver tissue, whereas TXNDC11 decreased the expression. . In addition, the present invention could be completed by confirming that an increase in the expression of TXNDC7 or a decrease in the expression of TXNDC11 in hepatocellular carcinoma is related to a poor prognosis of hepatocellular carcinoma.

As a prior art, Korean Patent Registration No.0877554 describes TXNDC11 as a marker for predicting the survival time of liver cancer patients, but 56 kinds of genes other than TXNDC are used as main markers, and TXNDC11 is included as an additional gene. The composition for diagnosing hepatocellular carcinoma including reduced expression of TXNDC11 and TXNDC7 in hepatocellular carcinoma is not described or suggested at all. Korean Patent Publication No. 2017-0021234 describes TXN as a biomarker for non-invasive diagnosis of liver cancer, but TXNDC7 and TXNDC11 of the present invention are not described.

Non-patent literature, Chen, D., et al., (2018) describes that TXNDC9 is increased in expression in hepatocellular carcinoma, and that this increase in expression is related to the progression of hepatocellular carcinoma. Increased expression of TXNDC7 and decreased expression of TXNDC11 of are not described and implied at all.

Korean Patent Registration No. 0877554, a marker for predicting the survival time of a liver cancer patient, a kit and a microarray including the same, and a method for predicting the survival time of a liver cancer patient using the marker, 2008. 12. 30. Registration. Korean Patent Application Publication No. 2017-0021234, a specific biomarker set for non-invasive diagnosis of liver cancer, 2017. 02. 27. Publication.

Hyun-Koo Woo, Research on genomic profiling of hepatocellular carcinoma, Molecular Cell Biology News, 2011. Chen, D., et al., TXNDC9 promotes hepatocellular carcinoma progression by positive regulation of MYC-mediated transcriptional network, Cell Death and Disease. 9, 1110, 2018. Clackson, T., et al., Making antibody fragments using phage display libraries, Nature, 352, 624-628, 1991. Horna-Terron, E., et al., TXNDC5, a newly discovered disulfied isomerase with a key role in cell physiology and pathology, Int. J. Mol. Sci., 15(12), 23501~23518, 2014. Kohler, G., et al., Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, European J Immunology, 6, 511-519, 1976. Lu, A., et al., TXNDC9 expression in colorectal cancer cells and its influence on colorectal cancer prognosis, Cancer Invest., 30(10), 721~726, 2012. Zhang, S.F., et al., TXNDC17 promotes paclitaxel resistance via inducing autophagy in ovarian cancer, Autophagy, 11(2), 225-238, 2015.

An object of the present invention is to provide a biomarker composition for diagnosing hepatocellular carcinoma or predicting prognosis comprising TXNDC7 (thioredoxin-domain-containing protein 7).

The present invention relates to a biomarker composition for diagnosing hepatocellular carcinoma (HCC) or predicting prognosis, comprising TXNDC7 (thioredoxin-domain containing protein 7) as an active ingredient.

The biomarker composition may include TXNDC7 and TXNDC11.

The TXNDC7 may have increased expression in hepatocellular carcinoma compared to normal liver tissue.

The TXNDC11 may have decreased expression in hepatocellular carcinoma compared to normal liver tissue.

In another aspect, the present invention relates to a kit for diagnosing hepatocellular carcinoma or predicting prognosis comprising a primer pair or probe that specifically binds to the TXNDC7 gene.

The kit for diagnosing hepatocellular carcinoma or predicting prognosis may include a primer pair or probe that specifically binds to the TXNDC7 and TXNDC11 genes.

In yet another aspect, the present invention relates to a kit for diagnosing hepatocellular carcinoma or predicting prognosis comprising an antibody that specifically binds to the TXNDC7 protein.

The antibody may be a monoclonal antibody (mAb) or a polyclonal antibody (pAb).

The present invention also provides a first step of measuring the expression level of the TXNDC7 gene or protein expressed in hepatocellular carcinoma of a hepatocellular carcinoma patient; A second step of comparing the expression level of the TXNDC7 gene or protein with a normal control sample; And a third step of determining that the TXNDC7 gene or protein expression level in the hepatocellular carcinoma is higher than that of the normal control sample as hepatocellular carcinoma. It relates to a method for providing information necessary for diagnosing HCC or predicting prognosis.

The method of providing information necessary for diagnosing hepatocellular carcinoma or predicting prognosis may further analyze the expression level of the TXNDC11 gene.

The expression of the TXNDC11 gene can be determined as hepatocellular carcinoma when the expression level in hepatocellular carcinoma is lower than that of a normal control sample.

The method of measuring the gene expression level is reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (competitive RT-PCR), real-time reverse transcription polymerase reaction (real-time RT-PCR). , RNase protection assay (RNase protection assay), Northern blot (Northern blot), it can be confirmed through one or more methods selected from the group consisting of a DNA chip (DNA chip).

The protein expression level can be measured through an antigen-antibody reaction.

The antigen-antibody reaction was determined by Western blot, immunohistochemical staining, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), radioimmunodiffusion, and immunoprecipitation assay. ), a complement fixation assay, and a protein chip, and at least one method selected from the group consisting of.

Hereinafter, the present invention will be described in detail.

The present invention relates to a biomarker composition for diagnosing hepatocellular carcinoma or predicting prognosis comprising TXNDC7 as an active ingredient.

In the present invention, "hepatocellular carcinoma (HCC)" refers to primary liver cancer derived from hepatocytes.

In the present invention, "prognosis" refers to the course of diseases such as relapse or recurrence of hepatocellular carcinoma, hepatocellular carcinoma-caused death or the likelihood of progression, including metastatic spread and drug resistance.

In the present invention, the term "prediction" means that the patient responds favorably or unfavorably to a therapy such as chemotherapy or radiation therapy, so that the patient is treated with treatment, for example, a specific therapeutic agent, and/or surgical removal of the primary tumor, And/or survival after treatment with chemotherapy for a certain period of time without recurrence of cancer and/or. The prediction method of the present invention can be used clinically by selecting and applying the most appropriate treatment regimen for any particular patient. The prediction method of the present invention is to determine whether a patient preferentially responds to a treatment regimen, such as a prescribed treatment regimen, including administration of a prescribed treatment or combination, surgical intervention, chemotherapy, etc. Can predict long-term survival or systemic or local recurrence. In addition, it is possible to plan to minimize unnecessary adjuvant chemotherapy or to use more effective adjuvant chemotherapy for patients with predicted systemic or local recurrence.

In the present invention, the term "biomarker" refers to a marker capable of distinguishing normal or pathological conditions or predicting a treatment response and measuring objectively, genes, proteins, such as DNA and RNA, Fats, metabolites, and the like are used to change their patterns.

In the present invention, "TXNDC (thioredoxin-domain containing protein)" is a regulator that regulates the oxidation-reduction (redox) state of cells, and various kinds of proteins such as redox homeostasis, oxidative stress, disulfide-containing proteins, and transcriptional regulation It is a factor involved in function. The TXNDC family is made up of 17 and regulates redox states at distinct intracellular locations including the plasma membrane, cytoplasm, endoplasmic reticulum and nucleus.

The biomarker composition may be a composition comprising all or part of the TXNDC7 gene sequence, or a composition comprising the TXNDC7 protein or an immunogenic fragment thereof.

The biomarker composition may additionally include all or part of the TXNDC11 gene sequence.

The expression of TXNDC7 is increased in hepatocellular carcinoma compared to normal liver tissue, while the expression of TXNDC11 is decreased in hepatocellular carcinoma compared to normal liver tissue.

In addition, the increase in the expression of TXNDC7 or the decrease in the expression of TXDNC11 is correlated with a decrease in the survival rate of HCC patients at each TNM stage of HCC, and is associated with a poor prognosis of HCC.

In the present invention, the "TNM stage" is a concept for classifying the progression of cancer, and is usually classified into 4 stages from stage 1 to stage 4, and the number increases as the degree of cancer progresses.

The increase in the expression of TXNDC7 may be an increase in the expression of genes and proteins.

The decrease in the expression of TXNDC11 may be a decrease in the expression of the gene.

The decrease in expression of the gene may be a decrease in mRNA expression.

In yet another aspect, the present invention relates to a kit for diagnosing hepatocellular carcinoma or predicting prognosis comprising a primer pair or probe that specifically binds to the TXNDC7 gene.

The kit for diagnosing or predicting the prognosis of hepatocellular carcinoma may additionally include a primer pair or a probe that specifically binds to the TXNDC11 gene.

Hepatocellular carcinoma diagnosis or prognosis prediction kit comprising a primer pair or probe that specifically binds to the TXNDC7 gene, or an additional primer pair or probe that specifically binds to the TXNDC11 gene for diagnosis or prognosis of hepatocellular carcinoma The kit is for detecting the TXNDC7 or TXNDC11 gene expressed in hepatocellular carcinoma, and the gene is preferably an mRNA.

In the present invention, "primer" is a nucleic acid sequence having a short free 3-terminal hydroxyl group (free 3'hydroxyl group) and can form a base pair with a complementary template, and the start for template strand copying Refers to a short nucleic acid sequence that acts as a point. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. PCR conditions, the length of the sense and antisense primers can be appropriately selected according to techniques known in the art.

The primer is a primer capable of amplifying all or part of the TXNDC7 or TXNDC11 gene sequence, and has a base sequence complementary to all or part of the TXNDC7 or TXNDC11 gene sequence, and a sense primer and an antisense primer are paired.

In the present invention, "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to several hundred bases capable of specifically binding to an mRNA, and is labeled The presence or absence and expression level can be checked. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. Selection of an appropriate probe and conditions for hybridization can be appropriately selected according to techniques known in the art.

The probe binds to all or part of the TXNDC7 or TXNDC11 gene sequence, and may have a base sequence complementary to all or part of the TXNDC7 or TXNDC11 gene sequence.

The kit for diagnosing or predicting HCC comprising a primer pair or probe that specifically binds to the TXNDC7 or TXNDC11 gene, in addition to the primer pair, is a test tube or other suitable container, reaction buffer, deoxyribonucleotide (dNTPs), DNA / RNA polymerase and enzymes such as reverse transcriptase, DNase, RNase inhibitors, sterile water, and the like.

In addition, the present invention relates to a kit for diagnosing hepatocellular carcinoma or predicting prognosis comprising an antibody (antibody, Ab) that specifically binds to the TXNDC7 protein.

The kit for diagnosing or predicting the prognosis of hepatocellular carcinoma comprising an antibody that specifically binds to TXNDC7 is for detecting the TXNDC7 protein expressed in hepatocellular carcinoma. It may include a secondary antibody labeled with a fluorescent material, a color developing substrate, and the like. In the above, a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. may be used as the substrate, and as a color developing enzyme, peroxidase ), alkaline phosphatase, etc. may be used. In addition, FITC, RITC, Cy3, Cy5, etc. may be used as the fluorescent material, and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenedianim), TMB (tetramethyl benzidine), and the like can be used.

In the present invention, "antibody (Ab)" refers to a specific protein molecule directed against an antigenic site. Preferably, the antibody refers to an antibody that specifically binds to the TXNDC7 protein or an immunogenic fragment (antigen) thereof, and comprises a monoclonal antibody (mAb), a polyclonal antibody (pAb), and a recombinant antibody. Can contain all. Producing the antibody can be easily prepared using techniques well known in the art. The polyclonal antibody can be obtained from serum collected after injection of the TXNDC7 protein or an immunogenic fragment thereof into an animal. Any animal host such as goat, rabbit, pig, and sheep can be used as the animal. The monoclonal antibody is a hybridoma method (Kohler, G. et al., 1976), or a phage antibody library (Clackson, T. et al., 1991) technology, as well known in the art to which the present invention belongs. It can be manufactured. In order to perform the hybridoma method, cells of an immunologically suitable host animal such as a mouse, and a cancer or myeloma cell line may be used. Thereafter, after fusion of these two types of cells by a method using polyethylene glycol, that is, a method well known in the art to which the present invention pertains, the antibody-producing cells can be proliferated by a standard tissue culture method. . Thereafter, after obtaining a homogeneous cell population by subcloning by a limited dilution technique, hybridomas capable of producing antibodies specific for the TXNDC7 protein of the present invention in vitro or in vivo according to standard techniques. Can be cultured in large quantities. The phage antibody library method obtains an antibody gene that specifically recognizes the TXNDC7 protein or immunogenic fragment thereof of the present invention, and expresses it in the form of a fusion protein on the surface of a phage to produce an antibody library in vitro. And, it can be carried out by separating and producing a monoclonal antibody that binds to the TXNDC7 protein of the present invention or an immunogenic fragment thereof from the library. Antibodies prepared by the above methods can be separated by methods such as electrophoresis, dialysis, ion exchange chromatography, and affinity chromatography.

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

A kit for diagnosing HCC or predicting prognosis including the antibody may include an aptamer, a peptide, or the like capable of specifically binding to the TXNDC7 protein instead of the antibody.

In the present invention, "aptamer" means a special kind of single-stranded nucleic acid (DNA, RNA, or modified) that has a stable tertiary structure by itself and can bind to a target molecule with high affinity and specificity. Nucleic acid) is a type of polynucleotide consisting of a polynucleotide that can specifically bind to an antigenic substance in the same way as an antibody, but has higher stability than protein, has a simple structure, and is composed of a polynucleotide that is easy to synthesize. It can be used in place of an antibody.

In the present invention, "peptide" has an advantage of high binding power to a target substance, and does not denature even during thermal/chemical treatment. Also, because the molecular size is small, it can be attached to other proteins and used as a fusion protein. Specifically, since it can be used by attaching it to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery material.

In another aspect, the present invention is a first step of measuring the expression level of the TXNDC7 gene or protein expressed in hepatocellular carcinoma of a hepatocellular carcinoma patient; A second step of comparing the expression level of the TXNDC7 gene or protein with a normal control sample; And a third step of determining that the TXNDC7 gene or protein expression level in the hepatocellular carcinoma is higher than that of the normal control sample as hepatocellular carcinoma. It relates to a method for providing information necessary for diagnosing HCC or predicting prognosis.

The method of providing information necessary for diagnosing hepatocellular carcinoma or predicting prognosis may further analyze the expression level of the TXNDC11 gene.

The expression of the TXNDC11 gene can be determined as hepatocellular carcinoma when the expression level in hepatocellular carcinoma is lower than that of a normal control sample.

The method of measuring the expression level of the TXNDC7 or TXNDC11 gene includes a reverse transcription polymerase chain reaction (RT-PCR), a competitive reverse transcription polymerase reaction (RT-PCR), and a real-time RT-PCR. -PCR), RNase protection assay (RNase protection assay), Northern blot (Northern blot) and may be one or more selected from the group consisting of a DNA chip (DNA chip), but is not limited thereto.

The TXNDC7 protein expression level can be measured through an antigen-antibody reaction.

The antigen-antibody reaction can be carried out according to various quantitative or qualitative immunoassay protocols previously developed. The immunoassay format is Western blot, immunohistochemical stain, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), radioimmunodiffusion, immunoprecipitation assay. , At least one selected from the group consisting of a complement fixation assay and a protein chip, but is not limited thereto.

The present invention relates to a biomarker composition for diagnosing hepatocellular carcinoma or predicting prognosis including TXNDC7, and as a result of analyzing the expression of the TXNDC family in hepatocellular carcinoma and normal liver tissue, the mRNA and protein of TXNDC7 in hepatocellular carcinoma compared to normal liver tissue While the expression was increased, it was confirmed that the expression of TXNDC11 decreased. In addition, it was confirmed that an increase in the expression of TXNDC7 or a decrease in the expression of TXNDC11 is associated with a poor prognosis of HCC.

Through this, TXNDC7 or TXNDC11 of the present invention can be used as a biomarker of hepatocellular carcinoma, and diagnosis and prognosis of hepatocellular carcinoma can be diagnosed and predicted through analysis of the expression level of TXNDC7 or TXNDC11 in hepatocellular carcinoma, and prevention or treatment of hepatocellular carcinoma In development, it is also expected to have high utilization value.

1 shows the results of analyzing the mRNA expression change (fold change) of TXNDC families in hepatocellular carcinoma compared to normal liver tissue.
2 is an immunohistostaining result confirming the protein expression of the TXNDC families in hepatocellular carcinoma, (A) is a result of comparing the protein expression between the TXNDC families, (B) is hepatocellular carcinoma compared to normal liver tissue (Normal) ( HCC) shows the results of comparing the protein expression of the TXNDC families.
3 shows the results of analyzing the correlation between the mRNA expression of the TXNDC family in HCC and the survival rate of HCC patients. Statistical significance is indicated when p<0.05.
Figure 4 shows the results of analyzing the correlation between the mRNA expression of the TXNDC family according to the TNM stage of the hepatocellular carcinoma patient and the survival rate of the hepatocellular carcinoma patient. Statistical significance is indicated when p<0.05.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the content introduced herein becomes thorough and complete, and is provided to sufficiently convey the spirit of the present invention to those skilled in the art.

< Example One. Hepatocellular carcinoma Secure patient clinical information and gene expression profile data>

For gene expression analysis, RNAseqV2-RSEM_gene information and clinical information, including gene expression profile data of 377 HCC samples, were obtained from Firebrowse (http://firebrowse.org/). Among them, clinical information and gene expression profile data of 367 hepatocellular carcinoma patients in Table 1, excluding 7 patients with hepatocholangiocarcinoma and 3 patients with fibrolamellar carcinoma, were used for analysis.

Hepatocellular carcinoma patients (n=367) Number of patients (persons) gender male 249 female 118 age ≤60 176 >60 190 TNM weapon Step I 171 Stage II 83 Stage III 85 IV stage 4 None 24 Histological grade
(histological grade) Rank 1 53 Grade 2 174 3 ratings 123 4 ratings 12 None 5 Life or death survival 236 Dead 131 Child taxonomy
(Child-Pugh classification) A 218 B 21 C One None 127 Ear fibrosis score
(Ishak fibrosis score) 0-no fibrosis 74 1,2-portal fibrosis 31 3,4-fiber septum 30 5- nodularity and incomplete cirrhosis 9 6-confirmed cirrhosis 72 None 151

< Example 2. Hepatocellular carcinoma In the patient TXNDC Families mRNA And protein expression confirmation>

Example 2-1. Confirmation of mRNA expression of TXNDC families

The information obtained by analyzing the mRNA expression levels of TXNDC families in HCC and normal liver tissues of 367 HCC patients of Example 1 was obtained from Firebrowse (http://firebrowse.org/viewGene.html?gene=thioredoxin). Then, the mRNA expression levels of the TXNDC families were compared and analyzed.

Specifically, the mRNA expression level in HCC compared to normal liver tissues of 16 genes excluding TXNDC8 among the 17 TXNDC families was compared, and the changed mRNA expression level was converted to a log value, and the amount of mRNA expression changed (fold change). And the results are shown in FIG. 1 and Table 2 below.

Gene name tinnitus Fold change
(log) TXNDC1 TMX1, thioredoxin-related transmembrane protein 1 0.9 TXNDC2 - 0.8 TXNDC3 NME8, NME/NM23-family member 8 0.9 TXNDC4 ERP44, endoplasmic reticulum protein 44 0.9 TXNDC5 - 1.0 TXNDC6 NME9, NME/NM23-family member 9 1.1 TXNDC7 PDIA6, protein disulfide isomerase family A member 6 1.3 TXNDC9 - 1.2 TXNDC10 TMX3, thioredoxin-related transmembrane protein 3 1.0 TXNDC11 - 0.7 TXNDC12 - 1.1 TXNDC13 TMX4, thioredoxin-related transmembrane protein 4 1.2 TXNDC14 TMX2, thioredoxin-related transmembrane protein 2 1.1 TXNDC15 - 1.0 TXNDC16 - 1.0 TXNDC17 - 1.0

As shown in the result of confirming the mRNA expression of the TXNDC family in hepatocellular carcinoma compared to the normal liver tissue of Fig. 1 and Table 2, the change amount of TXNDC7 was 1.3, and the expression level in hepatocellular carcinoma compared to normal liver tissue It was confirmed that the increase was confirmed, which was higher than the amount of change in the mRNA expression of TXNDC9, which is known to increase in hepatocellular carcinoma, of 1.2. On the other hand, the amount of change in the mRNA expression of TXNDC11 was 0.7, and it was confirmed that the expression was decreased in hepatocellular carcinoma compared to normal liver tissue.

Through this, in the case of TXNDC7 of the present invention, the mRNA expression level in HCC is increased compared to the normal liver tissue, and in the case of TXNDC11, the mRNA expression in HCC is decreased compared to the normal liver tissue. It was found that it was available as.

Example 2-2. Confirmation of protein expression of TXNDC families

In order to confirm the protein expression of the TXNDC families in hepatocellular carcinoma, immunohistostaining results related to the TXNDC families were obtained from the Human Protein Atlas portal (http://www.proteinatlas.org), and the results are shown in FIG. 2.

As shown in the immunohistostaining results confirming the protein expression of the TXNDC families in hepatocellular carcinoma of FIG. 2(A), it was confirmed that the protein expression appears different depending on the TXNDC families.

As shown in the result of comparing the protein expression of the TXNDC family in hepatocellular carcinoma (HCC) compared to normal liver tissue (N) of FIG. 2(B), TXNDC7 and TXNDC9 increased protein expression in hepatocellular carcinoma compared to normal liver tissue. Confirmed. In particular, as shown in (B) of Figure 2, by confirming that the TXNDC7 protein is more expressed in hepatocellular carcinoma than TXNDC9, it was found that TXNDC7 is a better diagnostic marker for hepatocellular carcinoma than TXNDC9, which is known as a hepatocellular carcinoma diagnostic marker.

<Example 3. Confirmation of correlation between expression of TXNDC family and prognosis of HCC>

Example 3-1. Analysis of correlation between mRNA expression and survival rate of TXNDC families

In order to confirm the correlation between the mRNA expression of the TXNDC family in HCC and the survival rate of HCC patients, clinical information and gene expression profile data of 367 HCC patients of Example 1 were used for analysis.

Accumulated mortality was calculated using the Kaplan-Meier method based on the time to the first event used as an outcome variable, and the survival period for statistical analysis was the difference between the operation day and the death date, and then 367 The cumulative overall survival curve according to the mRNA expression of HCC patients was analyzed, and the results are shown in FIG. 3.

The log-rank test was used to test the hypothesis to compare the survival distribution of the two samples in the overall survival cumulative curve, and the cutoff value of the mRNA expression data for the log-rank test was determined by Cutoff Finder (http://molpath). .charite.de/cutoff) was used. In addition, it was confirmed that the log-rank test shows statistical significance when p<0.05.

As shown in FIG. 3, it was confirmed that, as with TXNDC9, when the mRNA expression level of TXNDC7 is high, the survival rate of HCC patients is decreased, and the prognosis of HCC is poor due to a high risk of death (Hazard ratio, HR). On the other hand, it was confirmed that the higher the mRNA expression level of TXNDC11, the higher the survival rate of HCC patients and the lower the mortality risk, the better the prognosis for HCC.

Example 3-2. Expression-related survival rate analysis of TXNDC families according to TNM stage in HCC patients

The relationship between the mRNA expression of the TXNDC family and the prognosis of HCC was confirmed in the TNM stage of HCC patients.

Using the clinical information and gene expression profile data of 367 hepatocellular carcinoma patients of Example 1, the mRNA expression level of HCC patients by TNM stage and the level of mRNA expression at each stage of TNM using the same method as the total survival cumulative curve analysis of Example 3-1. The cumulative survival rate was analyzed, and the results are shown in FIG. 4.

As shown in FIG. 4, it was confirmed that the high mRNA expression of TXNDC7 decreased the survival rate of HCC patients at all TNM stages, thereby confirming that there was a significant correlation with the poor prognosis of HCC, and high mRNA expression of TXNDC11 It was confirmed that the survival rate of HCC patients at stages I and III-IV of the TNM stage was significantly associated with a good prognosis of HCC. On the other hand, in the case of TXNDC9, which is known to be available for diagnosis of hepatocellular carcinoma due to its increased expression in hepatocellular carcinoma, it was confirmed that there was a significant correlation only in stages III-IV of the TNM stage. It can be seen that TXNDC7 or TXNDC11 is more accurate than TXNDC9.

From the results of Examples 3-1 and 3-2, it was found that the high mRNA expression of TXNDC7 of the present invention is highly related to the poor prognosis of HCC, and the high mRNA expression of TXNDC11 is related to the good prognosis of HCC. It was found that TXNDC7 and TXNDC11 can be used as biomarkers for diagnosis and prognosis of hepatocellular carcinoma.

Claims (14)

A biomarker composition for diagnosing hepatocellular carcinoma (HCC) or predicting prognosis containing TXNDC7 (thioredoxin-domain containing protein 7) as an active ingredient. The method of claim 1,
The biomarker composition is a biomarker composition for diagnosing hepatocellular carcinoma or predicting prognosis, comprising TXNDC7 and TXNDC11. The method according to claim 1 or 2,
The TXNDC7 is a biomarker composition for diagnosing hepatocellular carcinoma or predicting prognosis, characterized in that the expression is increased in hepatocellular carcinoma compared with normal liver tissue. The method of claim 2,
The TXNDC11 is a biomarker composition for diagnosing hepatocellular carcinoma or predicting prognosis, characterized in that the expression is reduced in hepatocellular carcinoma compared to normal liver tissue. A kit for diagnosing hepatocellular carcinoma or predicting prognosis comprising a primer pair or a probe that specifically binds to the TXNDC7 gene of claim 1. The method of claim 5,
The kit for diagnosing or predicting the prognosis of hepatocellular carcinoma comprises a pair of primers or probes that specifically bind to TXNDC7 and TXNDC11 genes. A kit for diagnosing hepatocellular carcinoma or predicting prognosis comprising an antibody that specifically binds to the TXNDC7 protein of claim 1. The method of claim 7,
The antibody is a kit for diagnosing hepatocellular carcinoma or predicting prognosis, characterized in that the antibody is a monoclonal antibody (mAb) or a polyclonal antibody (pAb). 1 step of measuring the expression level of the TXNDC7 gene or protein expressed in hepatocellular carcinoma of the hepatocellular carcinoma patient;
A second step of comparing the expression level of the TXNDC7 gene or protein with a normal control sample; And
A third step of determining that the TXNDC7 gene or protein expression level in the HCC is higher than that of the normal control sample as HCC;
A method of providing information necessary for diagnosing hepatocellular carcinoma or predicting prognosis comprising a. The method of claim 9,
The method of providing information necessary for diagnosing HCC or predicting prognosis is a method of providing information necessary for diagnosing HCC or predicting prognosis, characterized in that the expression level of the TXNDC11 gene is additionally analyzed. The method of claim 10,
When the expression level of the TXNDC11 gene is lower than that of a normal control sample, the expression level of the TXNDC11 gene is determined to be hepatocellular carcinoma. The method of claim 9 or 10,
The method of measuring the gene expression level is reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (competitive RT-PCR), real-time reverse transcription polymerase reaction (real-time RT-PCR). , RNase protection assay (RNase protection assay), Northern blot (Northern blot) and DNA chip (DNA chip), characterized in that at least one selected from the group consisting of a method for providing information necessary for the diagnosis or prognosis of hepatocellular carcinoma. The method of claim 9,
The protein expression level is a method of providing information necessary for diagnosing hepatocellular carcinoma or predicting prognosis, characterized in that measured through an antigen-antibody reaction. The method of claim 13,
The antigen-antibody reaction was determined by Western blot, immunohistochemical staining, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), radioimmunodiffusion, and immunoprecipitation assay. ), a complement fixation assay, and a method for providing information necessary for diagnosing HCC or predicting prognosis, characterized in that measuring by at least one method selected from the group consisting of a protein chip.

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