KR102131519B1 - Biomarker for diagnosing of liver cancer metastasis or predicting prognosis of the same and use thereof - Google Patents

Abstract

The present invention relates to a biomarker for the diagnosis or prognosis of liver cancer metastasis and the use thereof, and more particularly, to a biomarker for the diagnosis or prognosis of liver cancer metastasis including miR-720 in exosomes and the use thereof.
The miRNA according to the present invention shows a specific expression pattern in patients with metastasis of liver cancer, and thus can be used as a biomarker for diagnosing liver cancer metastasis. In addition, since it can be used as a predictor for the prognosis of metastasis of liver cancer, it can be used for personalized medicine of liver cancer according to the determination of the treatment method and prediction of the prognosis of the patient.

Description

BIOMARKER FOR DIAGNOSING OF LIVER CANCER METASTASIS OR PREDICTING PROGNOSIS OF THE SAME AND USE THEREOF}

The present invention relates to a biomarker for the diagnosis or prognosis of liver cancer metastasis and its use, and more particularly, to a biomarker for the diagnosis or prognosis of liver cancer metastasis including miR-720 in exosomes and the use thereof.

Hepatocellular carcinoma (HCC) is the fifth most common tumor in the world, killing 500,000 people each year. In the meantime, treatment techniques of various techniques have been introduced into the clinic, but the survival rate of HCC patients is still poor, and the incidence of HCC in Korea has not improved over the past 20 years and has an incidence rate that is almost the same as the mortality rate. Chronic hepatitis B virus or hepatitis C virus infection and exposure to carcinogens such as alcohol and aflatoxin B1 are known to be major risk factors for HCC.

On the other hand, for diagnosis of hepatocellular carcinoma, imaging diagnostic tests such as ultrasound, CT, and MRI tests, serum tests (AFP, PIVKA-II), and pathological biopsy of biopsy materials are used. The serum AFP test shows a high value in approximately less than 70% of the patients, but it is often recognized that the high value of hardness is high even in patients with chronic liver disease, and the discrimination is important. On the other hand, in early hepatocellular carcinoma, it often shows a low value. Moreover, although the positive rate of PIVKA-II (protein induced by byitamin K antagonist-II) is less than 50%, it is known that the specificity to hepatocellular carcinoma is high, and the diagnostic accuracy is increased by the use of both. Pathological biopsy of biopsy material is an important test in the diagnosis of liver disease. However, it is sometimes difficult to identify cancer, particularly early hepatocellular carcinoma and non-cancerous tissue, solely by pathological features. In other words, micro lesions can be seen in regenerative nodules or early highly differentiated hepatocellular carcinoma. In particular, when the tumorous tissue is collected by a percutaneous needle biopsy, the collection amount may be limited, and more reliable diagnostic technology is required.

Republic of Korea Patent Publication No. 10-2018-0029936

The present invention provides a biomarker for the diagnosis or prognosis of liver cancer metastasis, including miR-720 in exosomes.

In addition, the present invention provides a composition for diagnosing or predicting liver cancer metastasis comprising an agent that measures the expression level of a biomarker for predicting or predicting liver cancer metastasis including miR-720 in exosomes.

In addition, the present invention provides a kit for diagnosing liver cancer metastasis or predicting prognosis.

In addition, the present invention provides a method for providing information for diagnosing liver cancer.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a biomarker (or biomarker composition) for diagnosis or prognosis of liver cancer metastasis, including miR-720 in exosomes.

The biomarker may be to diagnose extrahepatic metastasis of liver cancer or predict prognosis.

Since the biomarker of the present invention is excellent in accuracy and reliability as a marker for diagnosing liver cancer metastasis or predicting prognosis, it can be used for diagnosing metastasis of liver cancer or predicting prognosis.

In addition, the present invention provides a composition for diagnosing or predicting liver cancer metastasis comprising an agent that measures the expression level of a biomarker for predicting or predicting liver cancer metastasis including miR-720 in exosomes.

Unless otherwise specified in the present specification, the expression "measurement of the expression level of exosome miRNA" as used herein means detecting an object to be detected in the sample.

In the present invention, the target to be detected is miR-720 in the exosome present in the sample. That is, by detecting miR-720 in the exosome, it can be confirmed whether the liver cancer has metastasized.

The detection of exosome miRNA can be performed by extracting exosomes from a sample, and detecting miRNAs in the extracted exosomes. The detection of the exosome miRNA can be measured by a hybridization reaction and an amplification reaction, but is not limited thereto and can be easily performed using various techniques known in the art.

In addition, according to a preferred embodiment of the present invention, the expression level of the exosome miRNA can be measured from a biological sample of a patient suspected of metastasis of liver cancer.

The agent for measuring the expression level of the biomarker may include a primer or probe that specifically binds the biomarker.

Detection of exosome miRNA using primers can be performed by amplifying the gene sequence using an amplification method such as PCR and then confirming whether the gene is amplified by a method known in the art.

The primer is a nucleic acid sequence having a short free 3'hydroxyl group, capable of forming complementary templates and base pairs, and a short nucleic acid sequence serving as a starting point for template strand copying. Means Primers can be initiated by DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) at a suitable buffer and temperature and four different nucleoside triphosphates. In the present invention, it is possible to diagnose the onset of liver cancer by performing PCR amplification using sense and antisense primers that specifically bind to the miRNA to confirm the expression level. PCR conditions, sense and antisense primer lengths can be modified based on those known in the art.

The probe means a nucleic acid fragment such as RNA or DNA corresponding to several bases to tens of bases in short, and is labeled. The probe may be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, it is possible to diagnose liver cancer metastasis by performing hybridization using a probe complementary to the miRNA to confirm the expression level. The appropriate probe selection and hybridization conditions can be modified based on those known in the art.

Such primers or probes can be appropriately designed by those skilled in the art based on known sequences.

For example, primers or probes can be chemically synthesized using phosphoramidite solid support methods, or other well-known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (eg methyl phosphonate, phosphotriester, phosphoro Amidates, carbamates, etc.) or modifications to charged linkages (eg phosphorothioate, phosphorodithioate, etc.).

The expression level of the miRNA can be measured according to a method commonly used in the field of bio kits, such as reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerization. Enzyme reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting (Northern blotting) or genetic chip, and the like, but are not limited thereto.

In addition, according to another aspect of the present invention, there is provided a kit for diagnosing or predicting prognosis of liver cancer comprising the composition.

The kit may include an agent for measuring the expression level of the exosome miRNA, as well as tools, reagents, and the like commonly used in the art to be suitable for use as a kit for diagnosing or predicting the metastasis of liver cancer.

Examples of such tools or reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, a substrate capable of measuring enzyme activity and a reaction stopper may be included. The carrier is a soluble carrier, an insoluble carrier, and examples of the soluble carrier include physiologically acceptable buffers known in the art, for example, PBS, and examples of the insoluble carrier are polystyrene, polyethylene, polypropylene, polyester, poly Polymers such as acrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof.

For example, the diagnostic kit of the present invention may be a kit including essential elements necessary for performing RT-PCR. RT-PCR kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, in addition to each primer pair specific for the marker miRNA. The same enzyme, DNase, RNAse inhibitor, DEPC-water (DEPC-water), may include sterile water.

In addition, according to another aspect of the present invention, the present invention provides a method for providing information for diagnosis or prognosis of liver cancer metastasis, comprising the following steps: isolating exosomes from a biological sample of a subject (step a ); And comparing the expression level of miR-720 in the exosome with the expression level of the corresponding exosome miRNA in the control sample (step b).

In step b, when the expression level of miR-720 in the exosome is lower than that of the control group, the subject may be determined to be metastasis of liver cancer.

The term "low expression" used in referring to the expression level of the exosome miRNA herein refers to a healthy or normal individual or a subject to be compared when the biomarker indicates or is an abnormal process, disease or other condition in the individual. It refers to the value or level of the biomarker in the biological sample that is lower than the range or value of the biomarker detected in the obtained biological sample.

The term "biological sample" as used herein refers to any sample obtained from an individual whose expression of the biomarker of the present invention can be detected.

According to a preferred embodiment of the present invention, the biological sample may be selected from the group consisting of blood, serum, saliva, biopsy, blood, skin tissue, liquid culture, feces and urine, in particular Although not limited, it may be preferably serum, and may be prepared by treatment using a method commonly used in the art.

The miRNA according to the present invention shows a specific expression pattern in patients with metastasis of liver cancer, and thus can be used as a biomarker for diagnosing liver cancer metastasis. In addition, since it can be used as a predictor of the prognosis of metastasis of liver cancer, it can be used for personalized medicine of liver cancer according to the determination of the treatment method of the patient and the prediction of the prognosis. In addition, the present invention, by diagnosing metastasis or predicting the prognosis of liver cancer using the expression level of miR-720 in exosomes, it is possible to select individualized treatment methods for individuals with liver cancer in the same stage, thereby improving the quality of life of patients. It can contribute to the improvement of national health and health through improvement and increase in overall survival, and further contribute to the development of biomarker-based liver cancer standard treatment guidelines.

1 schematically shows the process of separating exosomes from serum.
Figure 2 schematically shows the process of isolating RNA (exosomal RNA) in exosomes.
Figure 3 shows the image confirmed by TEM exosomes (exosomes) separated in the serum (serum).
Figure 4 confirms the (exosomal) specific (specific) markers in the exosomes using a western blot (western blot).
Figure 5 shows the results of analyzing the expression level of miR-720 (exosomal miR-720) in exosomes present in serum.
Figure 6 shows the results of analyzing the expression level of miR-720 (exosomal miR-720) in exosomes according to the presence or absence of metastasis in the same patient.

Hereinafter, the present invention will be described in more detail through examples. The objects, features, and advantages of the present invention will be readily understood through the following examples. The present invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments introduced herein are provided to sufficiently convey the spirit of the present invention to those skilled in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples.

<Example>

Experiment content and method

I. Confirmation of exosomes

1. Blood separation

Serum was separated by centrifuging the patient's blood at 2000 RPM, 25° C. for 20 min. The separated serum was packaged in a dedicated tube at 440 ul and stored at -80°C.

2. Separation of exosomes

After exosome pellets were separated from the separated serum using exoquick (FIG. 1), resuspension was performed in 1 X phosphate buffered saline (PBS).

3. 1) Transmission electron microscope (TEM) analysis

After filtering the exosomes resuspensioned in 1X PBS, the TEM was measured.

3. 2) Western blot

After lysing the cells with RIPA buffer, debris were removed by centrifugation at 10000 RPM, 4° C. for 10 minutes, and then protein was quantified. After loading the sample on the SDS-PAGE gel, electrophoresis was performed for 90 hours and 1 hour and 30 minutes, followed by transfer to a polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK) (110 V 2h) ). The membrane was washed three times for 10 minutes using Tris-buffered saline and Tween 20 solution (TBS-T), and then washed with 10% skim milk. Blocked for 1 hour. Then, after attaching the primary antibody (1st Antibody), it was overnight incubated at 4° C. and washed 24 hours later. After tagged secondary antibodies (2 ^nd antibody) for 2 hours, then washed and were detected (detection) with ECL solution (solution).

Ⅱ. Confirmation of the amount of miRNA (exosomal miRNA) expression in exosomes

1. Blood separation and RNA (exosomal RNA) isolation in exosomes

The blood of the patient was centrifuged for 20 minutes at 2000RPM, 25°C, and serum was separated and packaged in a dedicated tube at 440 ul and stored at -80°C. Thereafter, RNA (exosomal RNA) in exosomes was isolated. The separation process is shown in FIG. 2.

2. cDNA synthesis

Exoquick is used to extract exosome RNA, and Taqman reverse transcription kit is used to obtain cDNA. PCR was performed using primers with sequences complementary to the sequence to be amplified (hsa-miR-720; 5'-UCUCGCUGGGGCCUCCA-3'). The necessary primers were purchased from Applied Biosystems and used. Master mix (100mM dNTP, Multiscribe Reverse Transcriptase (50U/ul), 10X Reverse transcription buffer, RNase inhibitor (20U/ul)) was added to 7ul, 5X RT primer 3ul and RNA sample 5ul were added to the mixture, and PCR was performed ( Table 1) PCR conditions were set to 16°C 30 minutes, 42°C 30 minutes, and 85°C 5 minutes (Table 2).

[Table 1]

[Table 2]

3. Quantitative Reverse-Transcription polymerase chain reaction

As a template, 0.5 ul of the hsa-miR-720 probe, a Taqman gene expression assay (20X), was reacted with 5 ul of the Taqman universal PCR Master mix (2X) using cDNA obtained through reverse transcription as a template, reacted cDNA reactant, and nuclease free water After adjusting the total 10ul capacity with free water), qRT-PCR was performed by dispensing into 384 wells (Table 3). The qRT-PCR conditions were set at 50°C 2 minutes, 95°C 10 minutes, and 40 cycles (95°C 15 seconds, 60°C 1 minute) (Table 4).

[Table 3]

[Table 4]

Experiment result

1. TEM image analysis

To confirm the exosomes present in the serum, the exosomes were separated using exoquick, and then visually confirmed whether the exosomes were correct using TEM (FIG. 3).

2. Western blot analysis result

It was proved that it was an exosome by confirming various specific markers in the exosome using a western blot (FIG. 4).

3. Results of miR-720 (exosomal miR-720 expression level) analysis in exosomes present in serum

According to the presence or absence of metastasis, the expression level of miR-720 (exosomal miR-720) in exosomes was confirmed. It was confirmed that the expression level of miR-720 (exosomal miR-720) in exosomes was reduced in the group with metastasis (HCC with mets(+)) compared to the group without metastasis (HCC without mets). Bar, it can be seen that exosome miR-720 (exosomal miR-720) is related to metastasis of liver cancer (FIG. 5).

4. Analysis of the expression level of miR-720 (exosomal miR-720) in exosomes according to the presence or absence of metastasis in the same patient

Expression level of miR-720 (exosomal miR-720) in exosomes with or without metastasis in the same patient to further confirm the relationship between miR-720 (exosomal miR-720) and metastasis in exosomes Was confirmed. As a result, it was confirmed that the expression level of miR-720 (exosomal miR-720) in exosomes was decreased in the group with metastasis (FIG. 6 ).

Claims (8)

delete delete A composition for diagnosing liver cancer metastasis comprising an agent that measures the expression level of a biomarker for diagnosing liver cancer metastasis including miR-720 in exosomes.
The method according to claim 3,
The composition for measuring liver cancer metastasis is characterized in that the agent for measuring the expression level of the biomarker comprises a primer or probe that specifically binds to the biomarker.
A kit for diagnosing liver cancer metastasis comprising the composition of claim 3 or 4.
A method of providing information for diagnosing liver cancer metastasis, including the following steps:
Isolating exosomes from the biological sample of the subject (step a); And
Comparing the expression level of miR-720 in the exosome with the expression level of the corresponding exosome miRNA in the control sample (step b).
The method according to claim 6,
In step b, if the expression level of miR-720 in the exosome is lower than that of the control group, the method is characterized in that the subject is determined to be liver cancer metastasis.
The method according to claim 6,
The biological sample is selected from the group consisting of blood, serum, saliva, biopsy, skin tissue, liquid culture, feces and urine.

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