KR20190090939A - Biomarker composition comprising human cytochrome P450 4A for diagnosis or predicting prognosis of hepatocellular carcinoma - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing or predicting prognosis of hepatocellular carcinoma, comprising human cytochrome P450 4A (CYP4A) as an active ingredient. More specifically, it has been confirmed that the gene and protein expression of human CYP4A in the liver tissue of the tumor part of hepatocellular carcinoma patients was significantly reduced compared to the liver tissue of a non-tumor part. As the stage of hepatocellular carcinoma progressed, the expression of human CYP4A decreased, and the lower the expression of human CYP4A, the higher the possibility of recurrence of hepatocellular carcinoma and the lower the patient survival rate. Therefore, human CYP4A is available as a biomarker of hepatocellular carcinoma, and the human CYP4A expression rate analysis is expected to be useful for the prevention or treatment of hepatocellular carcinoma by developing a composition for diagnosing or predicting prognosis of hepatocellular carcinoma.

Description

Biomarker composition comprising human cytochrome P450 4A for diagnosis or predicting prognosis of hepatocellular carcinoma}

The present invention relates to a biomarker composition for diagnosing or predicting hepatocellular carcinoma comprising human cytochrome P450 4A as an active ingredient.

Liver cancer is a generic term for all malignant tumors occurring in the liver. It is the fourth largest cancer death worldwide and is the leading cause of death among men in their 40s and 50s. These liver cancers are largely divided into metastatic liver cancer and primary liver cancer occurring in the liver. Among primary liver cancers, hepatocellular carcinoma derived from hepatocytes is the most common and is mainly called liver cancer (Kim Ji-hoon, 2013).

Hepatocellular carcinoma (HCC) is the third largest cancer in Korea and has a very poor prognosis with a 5-year survival rate of 9.6% (Bae, S.H., 2008). Hepatocellular carcinoma is known to cause various factors such as hepatitis virus, aflatoxin B1, drinking and diabetes, and the development of liver cancer is known to occur through multi-step processes such as genome and epigenetic mutation (Woo, 2011). .

Hepatocellular carcinoma develops rapidly in the 40s and peaks in the 60s. Hepatocellular carcinoma has few symptoms, and this is a period of active social activities, which is accompanied by fatigue, loss of appetite, and general weakness. (Kim Ji-hoon, 2013).

Despite recent developments in hepatocellular carcinoma and the development of methods of treatment, many patients are found to have advanced stages. Therefore, early diagnosis and curative treatment of liver cancer are the most effective methods for increasing the survival rate of patients.

Cytochrome P450 (CYP) is a prosthetic group that is a group of enzymes with heme. It is oxidative to various exogenous substances such as most drugs or environmental substances or endogenous substances such as steroids or lipids. It is known as a catalytic enzyme essential for life-giving metabolism. These cytochrome P450s are present in most organisms, from archaebacteria to humans, and to date more than 2,500 cytochrome P450 genes have been found (Guengerich, F.P., 2004).

There are 57 cytochrome P450 genes known in human genes, but about one-fourth of them have little known function, expression, or regulatory mechanism. In contrast, cytochrome P450 1A2 (CYP1A2) and 2A6 (CYP2A6) were found to play the most central role in carcinogenesis (Guengerich, F.P., 2003). CYP1A2 activates carcinogenicity of various heterocyclic amine compounds from foods such as charcoal meat, CYP2A6 activates carcinogenicity of nitrosamine compounds, and as CYP2A6 is expressed in the head or neck It has been shown to be highly correlated with developing cancer at these sites (Ding, X., et al., 2003). In addition, several studies have been conducted on the association between lung cancer and CYP2A6 associated with smoking in Japanese (Kamataki, T., et al., 2002).

Physiologically, the human cytochrome P450 4 family (CYP4) is associated with omega-hydroxylation of saturated branched chains and unsaturated fatty acids. CYP4A11, CYP4F2 and CYP4F3 have been studied the most in this CYP4 family, and it is known that they are highly expressed in human liver tissue (Johnson, A.L., et al., 2015). In addition, the human CYP4A subfamily consists of CYP4A11 and CYP4A22, which have very homologous gene sequences, and CYP4A22 is expressed at low levels in some tissues, and its activity is not yet known (Katheryne, ZE, et. al., 2013).

Accordingly, the present inventors, in the course of studying a biomarker for diagnosing or predicting hepatocellular carcinoma, the expression of human cytochrome P450 4A (CYP4A) gene and protein in the tumor part of the liver tissue of hepatocellular carcinoma patients is remarkably high. It was confirmed that the decrease, the lower the expression of human CYP4A, the lower the patient survival rate, the possibility of recurrence of hepatocellular carcinoma, and confirmed the progress of the cancer was able to complete the present invention.

In the prior art, US Patent Publication Nos. 2004-0248142 and 2014-0100127 disclose cytochrome P450 as a biomarker of hepatocellular carcinoma, which is similar to the configuration of the present invention, but the configuration thereof is different from that of human cytochrome P450 4A of the present invention. different.

Non-Patent Documents Meyer, K., et al. 2003. It is described that the gene expression of cytochrome P450 4A10 is reduced in hepatocellular carcinoma induced by genotoxic carcinogen, which is similar to the composition of the present invention, but CYP4A10 is cytochrome P450 of mouse and human of the present invention. The configuration is different from cytochrome P450 4A.

US Patent Publication No. 2004-0248142, Method for detecting hepatocellular carcinoma, published on December 09, 2004. United States Patent Application Publication No. 2014-0100127, Liver cancer diagnosis marker and use, published on April 10, 2014.

Kim Ji-hoon, Hepatocellular carcinoma, 2013 Fall meeting of Korean Internal Medicine, 2013. Hyun-Woo Woo, Genome Profiling of Hepatocellular Carcinoma, Molecular Cell Biology News, 2011. Bae, S. H., Up-to-date Information for Hepatocellular Carcinoma Treatment, J. Korean Med. Assoc., 51 (5), 457-474, 2008. Clackson, T., et al., Making antibody fragments using phage display libraries, Nature, 352, 624-628, 1991. Ding, X., et al., Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts, Annu. Rev. Pharmacol. Toxicol., 43, 149-173, 2003. Guengerich, F. P., Gytochrome P450, drugs, and diseases, Mol. Interv., 3, 194-204, 2003. Guengerich, F.P., Gytochrome P450: what have we learned and what are the future issues ?, Drug Metab. Rev., 36 (2), 159-197, 2004. Johnson, A.L., et al., Cytochrome P450 ω-Hydroxylase in Inflammation and Cancer, Adv. Pharmacol., 74, 223-262, 2015. Kamataki, T., et al., Role of human cytochrome P450 (CYP) in the metabolic activation of nitrosamine derivatives: application of genetically engineered Salmonella expressing human CYP, Drug Metab. Rev., 34, 667-676, 2002. Katheryne, Z.E., et al., CYP4 Enzymes As Potnetial Drug Targets: Focus on Enzyme Multiplicity, Inducers and Inhibitors, and Therapeutic Modulation of 20-hydroxyeicosatetraenoic Acid (20-HETE) Synthase and Fatty Acid ω-hydroxylase Activities, Curr. Top Med. Chem., 13 (12), 1429-1440, 2013. Kohler, G., et al., Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, European J Immunology, 6, 511-519, 1976. Meyer, K., et al., Molecular profiling of hepatocellular carcinomas developing spontaneously in acyl-CoA oxidase deficient mice: comparison with liver tumors induced in wild-type mice by a peroxisome proliferator and genotoxic carcinogen, Carcinogenesis, 24 (5), 975 -984, 2003.

An object of the present invention to provide a biomarker composition for diagnosing or prognostic hepatocellular carcinoma comprising human cytochrome P450 4A as an active ingredient.

The present invention relates to a biomarker composition for diagnosing or predicting hepatocellular carcinoma (HCC) comprising human cytochrome P450 4A (CYP4A) as an active ingredient.

The human CYP4A may be CYP4A11 and CYP4A22.

The human CYP4A may be reduced in expression according to progression of hepatocellular carcinoma or hepatocellular carcinoma compared to normal liver tissue.

The present invention also relates to a kit for diagnosing or predicting hepatocellular carcinoma comprising an antibody specifically binding to the human CYP4A.

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

The present invention also relates to a kit for diagnosing or predicting hepatocellular carcinoma comprising a primer pair or a probe specifically binding to the human CYP4A gene.

In addition, the present invention comprises the steps of measuring the human CYP4A protein or gene expression level expressed in liver tissue or hepatocytes of hepatocellular carcinoma patients; Comparing the human CYP4A protein or gene expression level with a normal control sample; And, if the human CYP4A protein or gene expression level in the liver tissue or hepatocytes of the patient is lower than the normal control sample 3 step to determine the hepatocellular carcinoma; Hepatocellular carcinoma comprising a method for providing information necessary for the diagnosis or prognosis It is about.

The human CYP4A protein expression level can be measured via an antigen-antibody response.

The antigen-antibody reactions include Western blot, immunohistochemical staining, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), radioimmunodiffusion, immunoprecipitation assay ), Complement fixation assay (complement fixation assay) and protein chips (protein chip) can be confirmed by one or more methods selected from the group consisting of.

The method for measuring human CYP4A gene expression level is reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real time RT-PCR (realtime RT-PCR) , RNase protection assay (RNase protection assay), northern blot (northern blot) and DNA chip (DNA chip) may be one or more 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 or predicting hepatocellular carcinoma (HCC) comprising human cytochrome P450 4A (CYP4A) as an active ingredient.

"Hepatocellular carcinoma" means primary liver cancer derived from hepatocytes.

The "prognosis" refers to the progress and cure of a disease, such as the possibility of relapse or recurrence of hepatocellular carcinoma, metastatic proliferation, and the possibility of hepatocellular carcinoma-causing death or progression, including drug resistance.

The term "prediction" means that the patient responds favorably or unfavorably to treatments such as chemotherapy or radiation therapy so that the patient is treated, for example, by surgical removal of certain therapeutic agents, and / or primary tumors, and / or cancer Survival and / or likelihood after treatment with chemotherapy for a certain period of time without recurrence. The prediction method of the present invention can be used clinically by selecting and applying the most appropriate treatment regimen for any particular patient. Prediction methods of the present invention determine whether a patient responds favorably to a treatment such as a prescribed treatment regimen, including, for example, administration of a predetermined treatment or combination, surgical intervention, chemotherapy, or the like, or after a treatment regimen. Long term survival or systemic or local recurrence is predictable. It can also be planned to minimize unnecessary adjuvant chemotherapy or to use adjuvant chemotherapy for patients who are expected to have systemic or local recurrence.

The "biomarker" refers to a marker that can distinguish a normal or pathological condition or predict a therapeutic response, and can be objectively measured. Genes, proteins, lipids, metabolites such as DNA and RNA And the like and variations of the pattern are used.

The biomarker composition may be a composition comprising a human CYP4A protein or an immunogenic fragment thereof or a composition comprising all or part of a human CYP4A gene sequence.

The human CYP4A is one of the CYP4 families, which may be CYP4A11 and CYP4A22. Preferably CYP4A11.

The human CYP4A is reduced in hepatocellular carcinoma compared to normal liver tissue. In addition, as stages of hepatocellular carcinoma progress, expression of human CYP4A in hepatocellular carcinoma gradually decreases.

The "stage" is a concept of classifying cancer progression, and is usually classified into four stages from stage 1 to stage 4, and the number increases as the cancer progresses.

The decrease in human CYP4A expression may be a decrease in expression of proteins and genes, and the decrease in human CYP4A gene expression may be a decrease in mRNA expression.

The present invention also relates to a kit for diagnosing or predicting hepatocellular carcinoma comprising a antibody or a primer pair or a probe specifically binding to a human CYP4A gene.

Hepatocellular carcinoma diagnostic or prognostic kit comprising the antibody specifically binding to human CYP4A is for detecting human CYP4A protein expressed in hepatocellular carcinoma, the substrate, suitable buffer, color development for immunological detection of the antibody Secondary antibodies labeled with enzymes or fluorescent materials, chromogenic substrates, and the like. As the substrate, a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, a slide glass made of glass, etc. may be used, and peroxidase (peroxidase) may be used. ), Alkaline phosphatase and the like can be used. In addition, FITC, RITC, Cy3, Cy5, etc. may be used as the fluorescent substance, and ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid)) and OPD as a chromogenic substrate (o-phenylenedia), TMB (tetramethyl benzidine) and the like can be used.

The term "antibody" refers to a specific protein molecule directed against an antigenic site. Preferably, the antibody refers to an antibody that specifically binds to a human CYP4A protein or an immunogenic fragment thereof, and includes all monoclonal antibodies (mAb), polyclonal antibodies (pAb), and recombinant antibodies. can do. Producing such antibodies can be readily prepared using techniques well known in the art. The polyclonal antibody can be obtained from serum collected after injecting a human CYP4A protein or an immunogenic fragment thereof (antigen) into an animal. The animal may be any animal host such as goat, rabbit, pig or sheep. The monoclonal antibody, as is well known in the art to which the present invention pertains, uses hybridoma method (Kohler, G. et al., 1976), or phage antibody library (Clackson, T. et al., 1991) technology. It can manufacture. In order to perform the hybridoma method, cells of an immunologically suitable host animal such as a mouse and cancer or myeloma cell line may be used. Thereafter, by using a method such as polyethylene glycol, that is, a method well known in the art to which the present invention belongs, after fusing these two kinds of cells, the antibody producing cells can be propagated by a standard tissue culture method. have. Subsequently, after obtaining a uniform cell population by subcloning by the limited dilution technique, hybridomas capable of producing antibodies specific for the human CYP4A protein of the present invention are in vitro or in accordance with standard techniques. It can be mass culture in vivo. The phage antibody library method obtains an antibody gene that specifically recognizes a human CYP4A protein or immunogenic fragment thereof of the present invention, and expresses it in the form of a fusion protein on the surface of the phage to express the antibody library in vitro. And a monoclonal antibody which binds to the human CYP4A protein of the present invention or an immunogenic fragment thereof from the library can be isolated and produced. Antibodies prepared by the above methods can be separated by electrophoresis, dialysis, ion exchange chromatography, affinity chromatography and the like.

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

Hepatocellular carcinoma diagnostic or prognostic kit comprising the antibody may include an aptamer, a peptide, or the like that can specifically bind to human CYP4A protein instead of the antibody.

The "aptamer" refers to a polynucleotide composed of a special kind of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure and having a characteristic of binding to a target molecule with high affinity and specificity. In one kind, aptamers can be used in place of antibodies because they are specifically composed of polynucleotides that can bind specifically to antigenic substances like antibodies, but are more stable than proteins, simple in structure, and easy to synthesize. .

The "peptide" has a high binding strength to the target material, and does not occur denaturation even during thermal / chemical treatment. In addition, the small size of the molecule can be used as a fusion protein by attaching to other proteins. Specifically, since it can be used by attaching to a polymer protein chain, it can be used as a diagnostic kit and drug delivery material.

Hepatocellular carcinoma diagnosis or prognostic kit comprising a primer pair or probe specifically binding to the human CYP4A gene is for detecting a human CYP4A gene expressed in hepatocellular carcinoma, the gene is human CYP4A mRNA.

The "primer" is a nucleic acid sequence having a short free 3-terminal hydroxyl group, which forms a base pair with a complementary template and serves as a starting point for template strand copying. Say. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. PCR conditions, sense and antisense primer lengths may be appropriately selected according to techniques known in the art.

The primer is a primer capable of amplifying all or part of a human CYP4A gene sequence. The primer has a base sequence complementary to all or a part of the human CYP4A gene sequence, and a sense primer and an antisense primer are paired.

The "probe" refers to nucleic acid fragments such as RNA or DNA, which are short to several bases to hundreds of bases that can specifically bind to mRNA, and are labeled to identify the presence or absence of expression of a specific mRNA. Can be. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. Selection of appropriate probes and hybridization conditions may be appropriately selected according to techniques known in the art.

The probe binds to all or a portion of the human CYP4A gene sequence, and may have a base sequence complementary to all or a portion of the human CYP4A gene sequence.

Hepatocellular carcinoma diagnosis or prognosis kit comprising a primer pair or probe specifically binding to the human CYP4A gene, in addition to the primer pair, a test tube or other suitable container, reaction buffer, deoxyribonucleotide (dNTPs), enzymes such as DNA / RNA polymerase and reverse transcriptase, DNase, RNase inhibitors, sterile water and the like.

The present invention also comprises the steps of measuring the human CYP4A protein or gene expression level expressed in liver tissue or hepatocytes of hepatocellular carcinoma patients; Comparing the human CYP4A protein or gene expression level with a normal control sample; And, if the human CYP4A protein or gene expression level in the liver tissue or hepatocytes of the patient is lower than the normal control sample 3 step to determine the hepatocellular carcinoma; Hepatocellular carcinoma comprising a method for providing information necessary for the diagnosis or prognosis It is about.

The human CYP4A protein expression level can be measured via an antigen-antibody response.

The antigen-antibody response can be carried out according to various quantitative or qualitative immunoassay protocols developed in the past. The immunoassay format is Western blot, immunohistochemical staining, 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 not limited thereto.

The method for measuring human CYP4A gene expression level is reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCT, real time RT-PCR (realtime RT-PCR) , RNase protection assay (RNase protection assay), northern blot (northern blot) and DNA chip (DNA chip) may be one or more selected from the group consisting of, but is not limited thereto.

The present invention relates to a biomarker composition for the diagnosis or prognosis of hepatocellular carcinoma comprising human cytochrome P450 4A (CYP4A) as an active ingredient, wherein the gene and protein expression of human CYP4A is non-tumor in liver tissue of the tumor part of hepatocellular carcinoma patient. It was confirmed that the hepatocellular carcinoma was significantly reduced compared with the partial hepatocellular carcinoma. As the stage of the hepatocellular carcinoma progressed, the expression of human CYP4A decreased, and the lower the expression of human CYP4A, the higher the possibility of recurrence of hepatocellular carcinoma, and the lower the patient survival rate. .

Accordingly, the human CYP4A of the present invention can be used as a biomarker of hepatocellular carcinoma, and the diagnosis and prognosis of hepatocellular carcinoma can be predicted through the analysis of the human CYP4A expression rate, and the value of the CYP4A can be high in the development of a preventive or therapeutic agent for hepatocellular carcinoma. It is expected to be.

1 is a result of analysis of human CYP4A protein expression in liver tissue of immunohistochemically stained hepatocellular carcinoma patients, (A) is a stained tissue tumor portion (HCC) and non-tumor portion of liver tissue of hepatocellular carcinoma patients The degree of human CYP4A expression in (non-tumor) and (B) shows the result of comparing the protein expression levels of human CYP4A in tumor and non-tumor areas by analyzing the degree of staining.
Figure 2 is a Western blot confirming the expression level of human CYP4A protein in liver tissue of hepatocellular carcinoma patients, (A) Western blot band confirming the human CYP4A expression in the tumor (T) and non-tumor (N) As a result, (B) shows the result of comparing the expression level of human CYP4A protein in the tumor part (HCC) and the non-tumor part by analyzing the band obtained from the western blot result.
Figure 3 shows the survival curves of hepatocellular carcinoma patients according to the expression level of human CYP4A protein, (A) human CYP4A protein expression and cumulative disease-free Survival, (B) human Correlation between CYP4A protein expression and Cumulative Overall Survival is shown.
FIG. 4 shows the results of confirming the expression level of human CYP4A mRNA in liver tissues of HCC and non-tumor liver tissues of HCC patients by real-time RT-PCR.
5 is a result of analyzing the degree of human CYP4A mRNA expression according to the progression of hepatocellular carcinoma, (A) according to the histological grade, (B) shows the result of analyzing the degree of human CYP4A mRNA expression according to the pathological stage Giving.

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 information provided herein is to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

<Example 1. Clinical information and liver tissue secured in patients with hepatocellular carcinoma>

The liver tissues of HCC patients were searched for biomarkers for diagnosis or prognosis of HCC.

Liver tissues of hepatocellular carcinoma patients were used from 1999 to 2013 in 120 patients who underwent hepatocellular carcinoma resection at Chungnam National University Hospital. In the case of liver tissue, it was fixed with paraffin and then H & E stained (hematoxylin and eosin stain), and the tumor portion and the non-tumor portion were selected on the tissue slide.

The pathological stage for hepatocellular carcinoma was a tumor, node and metastasis (TNM) stage, which was determined according to the 8th edition of the American Joint Committee on Cancer (AJCC) based on the timing of resection surgery. In addition, the clinical information of the patient is secured through the electronic medical records KHK and HSE are summarized in Table 1 below.

Hepatocellular Carcinoma Patients (n = 120) Number of patients (%) age ≤57 58 (48.3) > 57 62 (51.7) gender male 89 (74.2) female 31 (25.8) Whether you have HBV or HCV Uninfected 24 (20.0) infection 96 (80.0) Cirrhosis Nasal Cirrhosis 28 (23.3) Cirrhosis 92 (76.7) Pathological staging
(pathological stage,
TNM stage) I stage 31 (25.8) Stage II 79 (65.8) Stage III 8 (6.7) IV stage 2 (1.7) Histological grade
(histological grade) 1 rating (G1) 25 (20.8) 2 star (G2) 59 (49.2) 3 star (G3) 36 (30.0) 4 star (G4) 0 (0.0)

< Example  2. Hepatocellular Carcinoma  Determination of Human Cytochrome P450 4A (CYP4A) Protein Expression by Patient's Liver Staining>

Example 2-1. Confirmation of Human CYP4A Expression by Tissue Staining

Immunohistochemical staining was performed by treating human CYP4A-recognized antibodies on liver tissue slides of hepatocellular carcinoma patients obtained in Example 1, and performing digital scan of tissue files and ScanScope program (Aperio ScanScope CS system, Vista, USA) was analyzed for the degree of staining, the results are shown in FIG. At this time, the antibody that recognizes human CYP4A (Clone # EPR8276, Abcam, UK) used an antibody that can detect CYP4A11 and CYP4A22 at the same time, and divided the liver tissue of hepatocellular carcinoma patients into tumor and non-tumor parts and human CYP4A The expression level of was compared.

As shown in the results of staining the liver tissue of the hepatocellular carcinoma patient of Figure 1 (A) using human CYP4A antibody, human CYP4A in the non-tumor region (Non-Tumor) compared to the tumor region (HCC) despite the same liver tissue It was confirmed that a lot of expression of the deeply stained brown. Even when the asterisk portion marked on the tumor part and the non-tumor part was enlarged (figure below in (A)), the tumor part was almost brown, whereas the non-tumor part was mostly brown. It was.

In addition, the results of quantifying the expression level of human CYP4A in the non-tumor portion and the tumor portion of the hepatocellular carcinoma patient of FIG. 1 (B) showed that the expression of human CYP4A was significantly reduced in the tumor portion as compared to the non-tumor portion. It was.

Example 2-2. Western blots confirm expression

To more accurately confirm human CYP4A protein expression in liver tissue of hepatocellular carcinoma patients, 15 tissues from the patient tissues of Example 1 were selected.

From 15 tissues selected, tissues of the tumor part (HCC) and non-tumor part (Non-Tumor) at least 5 cm away from the tumor part were collected, homogenized using a homogenizer, and proteins were separated. Western blot was performed using the results, and the results are shown in FIG. 2. In this case, β-actin antibody (sc-1616, Santa Cruz Biotechnology, USA) for detecting β-actin was used as the antibody used in Example 2-1 and the loading control to detect human CYP4A.

As shown in FIG. 2, the expression of human CYP4A in the liver tissues of 15 hepatocellular carcinoma patients showed that the human CYP4A bands of the tumor parts (HCC, T) were non-tumor parts (Non-Tumor, N) of human CYP4A. Compared with the band, it was confirmed that it appeared to be thin or thin (A), and when it was numerically represented as a graph (B), it was confirmed that the expression level of human CYP4A in the tumor part was reduced compared to the non-tumor part.

Therefore, the results of Examples 2-1 and 2-2 showed that the expression of human CYP4A is reduced in the tumor part of hepatocellular carcinoma patients, which is a biomarker for diagnosing hepatocellular carcinoma. It is believed to show that it is available.

Example 2-3. Correlation of Pathologic Characteristics and Human CYP4A Expression in Hepatocellular Carcinoma Patients.

In order to confirm the relationship between the expression level of human CYP4A in liver tissue of hepatocellular carcinoma patients and the clinical pathologic characteristics of the patient, the clinical information of the hepatocellular carcinoma patient of Example 1 and the hepatocellular carcinoma patient of Example 2-1 The amount of human CYP4A expression analyzed from liver tissues is summarized in Table 2 below. In addition, through the post-treatment test of hepatocellular carcinoma patients, the survival rate of hepatocellular carcinoma patients according to human CYP4A expression was analyzed and the results are shown in FIG. 3.

Hepatocellular Carcinoma Patients (n = 120) Human CYP4A Expression lowness(%) height(%) 67 people 53 people age ≤57 30 (44.8) 28 (52.8) > 57 37 (55.2) 25 (47.2) gender male 49 (73.1) 40 (75.5) female 18 (26.9) 13 (24.5) Whether you have HBV or HCV Uninfected 14 (20.9) 10 (18.9) infection 53 (79.1) 43 (81.1) Cirrhosis Nasal Cirrhosis 17 (25.4) 11 (20.8) Cirrhosis 50 (74.6) 42 (79.2) Pathological staging
(pathological stage,
TNM stage) I stage 12 (17.9) 19 (35.8) Stage II 47 (70.1) 35 (60.4) Stage III 7 (10.4) 1 (1.9) IV stage 1 (1.5) 1 (1.9) Histological grade
(histological grade) 1 rating (G1) 8 (11.9) 17 (32.1) 2 star (G2) 33 (49.3) 26 (49.1) 3 star (G3) 26 (38.8) 10 (18.9) 4 star (G4) 0 (0.0) 0 (0.0)

As shown in Table 2, the relationship between the clinicopathologic characteristics of human hepatocellular carcinoma patients and human CYP4A expression levels was not correlated with human CYP4A expression levels in patients with age, sex, HBV or HCV infection, and liver cirrhosis. On the other hand, the relationship between the pathological stage and histological grades related to the progression of the carcinoma shows that in patients with low stage or histological grade, the proportion of patients with high expression level of human CYP4A is higher than that of patients with low expression level. On the other hand, as the stage or histological grade was higher, the proportion of patients with low expression level was higher than that of patients with high expression level of human CYP4A.

In addition, as shown in the results of survival analysis of hepatocellular carcinoma patients according to the expression of human CYP4A in FIG. 3, cumulative disease-free survival (A) and cumulative overall survival (A) after hepatocellular carcinoma treatment ( B) In all, it was confirmed that the survival rate of patients with high expression of human CYP4A was higher than that of patients with low expression of human CYP4A.

On the other hand, although not shown in the present specification, when analyzing the survival rate of hepatocellular carcinoma patients according to the expression level of CYP4F2, one of the CYP4 family expressed in human liver tissue, there is a big difference in the survival rate of patients according to the expression level of CYP4F2 It was confirmed that there was no.

Through this, the expression of human CYP4A decreases as the stage of hepatocellular carcinoma progresses, and it can be seen that it significantly affects the survival rate of patients, and human CYP4A can be used as a biomarker for predicting the progression and prognosis of hepatocellular carcinoma. I could see that.

Example 3 Analysis of Human CYP4A mRNA Expression in Hepatocellular Carcinoma Patients

Example 3-1. Expression of Human CYP4A mRNA by Real-Time RT-PCR

RNA was isolated and purified from liver tissue of the hepatocellular carcinoma patient obtained in Example 1, and mRNA expression of human CYP4A was analyzed.

Total RNA was isolated from the liver tissue of 20 hepatocellular carcinoma patients using a company-supplied manual using TRIzol reagent (Thermo Fisher Scientific, USA). At this time, liver tissue was divided into tumor parts and non-tumor parts to separate total RNA. Complementary DNA (cDNA) was synthesized using the isolated total RNA and the cDNA synthesis kit (amfiRivert cDNA synthesis master mix) of GenDEPOT. Using synthetic cDNA as a template, real-time reverse transcriptase polymerase reaction was performed using the primers of Table 3 and SYBR Green Real-time PCR master Mix (Toyobo, Japan) to confirm the expression level of human CYP4A mRNA. 4 is shown. At this time, β-actin (β-actin) was used as a quantitative control for mRNA expression analysis.

Gene Primer type Primer Sequence CYP4A Forward 5-CTCAACACAGCCACGCTTTC-3 Reverse 5-ACAAGTCGTGCAATGGGGAT-3 β-actin Forward 5-AGCGAGCATCCCCCAAAGTT-3 Reverse 5-GGGCACGAAGGCTCATCATT-3

As shown in FIG. 4, a comparison of the amount of human CYP4A mRNA expression in the non-tumor portion and the tumor portion (HCC) of hepatocellular carcinoma patients showed that the amount of human CYP4A mRNA expression in the tumor portion was significantly higher than that in the non-tumor portion. It was confirmed that it was reduced.

Example 3-2. Analysis of Human CYP4A mRNA Expression Using Hepatocellular Carcinoma Cohort Information

To determine the relationship between human CYP4A mRNA expression and hepatocellular carcinoma, 367 patients with hepatocellular carcinoma of Table 4 from Liver hepatocellular carcinoma (LIHC), a cohort of FIREHOSE (https://gdac.broadinstitute.org) Information was obtained and patient's TCGA RNA-seq data was obtained from the TCGA Research Network. The obtained TCGA RNA-seq data was normalized using the Rank Normalized module of GenePattern, and then analyzed the correlation between the clinical information of hepatocellular carcinoma patients and the expression level of human CYP4A mRNA, and the results are shown in FIG. 5. .

Patients enrolled in hepatocellular carcinoma cohort (n = 367) Number of patients age ≤60 176 > 60 190 None One gender male 249 female 118 Isaac Fibrosis Score
Ishak Fibrosis score No 0-fibrosis
(no fiborsis) 74 1,2-contextual fibrosis
(portal fibrosis) 31 3, 4-fiber diaphragm
(fibrous septa) 30 5-nodule formation and incomplete cirrhosis
(nodula formation and incomplete cirrhosis) 9 6-Cirrhosis
(established cirrhosis) 72 None 151 Pathological staging
(pathological stage,
TNM stage) I stage 171 Stage II 83 Stage III 85 IV stage 4 None 24 Histological grade
(histological grade) 1 rating (G1) 53 2 star (G2) 174 3 star (G3) 123 4 star (G4) 12 None 5 Survival survival 236 death 131

As shown in Figure 5, it was confirmed that the mRNA expression level of human CYP4A decreases as the histological grade (A) and the pathological stage (TNM stage) (B) of hepatocellular carcinoma patients progress.

Through the results of Example 3-1 and Example 3-2, it was confirmed that the mRNA expression level of human CYP4A is reduced as the stage and histologic grade of hepatocellular carcinoma progresses. It was found that it can be used as a biomarker for prognostic prediction.

Claims (10)

Biomarker composition for diagnosis or prognosis of hepatocellular carcinoma (HCC) comprising human cytochrome P450 4A (Cytochrome P450 4A, CYP4A) as an active ingredient. The method of claim 1,
The human CYP4A is CYP4A11 and CYP4A22 biomarker composition for diagnosing or predicting hepatocellular carcinoma. The method according to claim 1 or 2,
The human CYP4A is a biomarker composition for diagnosing or predicting hepatocellular carcinoma, characterized in that the expression is reduced in hepatocellular carcinoma compared to normal liver tissue. A kit for diagnosing or predicting hepatocellular carcinoma comprising an antibody that specifically binds to human CYP4A of claim 1. The method of claim 4, wherein
Said antibody is a monoclonal antibody (monoclonal antibody, mAb) or polyclonal antibody (polyclonal antibody, pAb), characterized in that hepatocellular carcinoma diagnosis or prognosis kit. A kit for diagnosing or predicting hepatocellular carcinoma comprising a primer pair or a probe specifically binding to the human CYP4A gene of claim 1. Measuring a human CYP4A protein or gene expression level expressed in liver tissue or hepatocytes of a hepatocellular carcinoma patient;
Comparing the human CYP4A protein or gene expression level with a normal control sample; And
Step 3 judging that the human CYP4A protein or gene expression level in the liver tissue or hepatocytes of the patient is lower than the normal control sample, hepatocellular carcinoma;
Method for providing information necessary for the diagnosis or prognosis of hepatocellular carcinoma comprising a. The method of claim 7, wherein
The human CYP4A protein expression level is measured by an antigen-antibody response to provide information necessary for diagnosing or predicting hepatocellular carcinoma. The method of claim 8,
The antigen-antibody reaction may include Western blot, immunohistochemical staining, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), radioimmunodiffusion, immunoprecipitation assay ), Complement fixation assay (complement fixation assay) and protein chip (protein chip) is a method for providing information necessary for the diagnosis or prognosis of hepatocellular carcinoma, characterized in that at least one method selected from the group consisting of. The method of claim 7, wherein
The method for measuring human CYP4A gene expression level is reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCT, real time RT-PCR (realtime RT-PCR) And RNase protection assay (RNase protection assay), Northern blot (northern blot) and DNA chip (DNA chip) is one or more selected from the group consisting of a method for providing information necessary for diagnosing or predicting hepatocellular carcinoma.

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