KR101987358B1 - Novel Biomarkers For Diagnosing Liver Cancer and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel liver cancer biomarker and a use thereof. More particularly, the present invention relates to a novel liver cancer biomarker and a use thereof. More particularly, the present invention relates to a novel liver cancer biomarker, A novel liver cancer biomarker comprising at least one exosome miRNA selected from the group consisting of Mir-150-5p, Mir-342-3p, Mir-16-5p, Mir-26b-5p and Mir- ; A composition for diagnosing liver cancer comprising the biomarker; And a kit for diagnosing liver cancer comprising the biomarker.

Description

[0001] The present invention relates to novel biomarkers for diagnosing liver cancer,

The present invention relates to novel liver cancer biomarkers and their uses, and more particularly to liver cancer biomarkers comprising exosome miRNA and uses thereof.

The diagnosis of hepatocellular carcinoma (HCC) is made through biopsy, and patients with chronic hepatitis and cirrhosis are at high risk for developing liver cancer, so surveillancetest is recommended for the early diagnosis of liver cancer. Hepatocellular carcinoma biomarkers or tumor markers are the factors that can help diagnosis and prognosis of liver cancer through blood test. Currently, serum alpha-fetoprotein is the most widely used blood biomarker or tumor marker of HCC. However, serum alpha-fetoprotein is elevated in only about 40-60% of patients with hepatocellular carcinoma, and the positive rate of hepatocellular carcinoma is significantly reduced. In addition, serum fetal protein is also a false positive that appears to be positive in chronic hepatitis without hepatocellular carcinoma or chronic liver disease such as cirrhosis.

It is known that microRNAs composed of 22 nucleotides are non-coding RNAs, but they do not produce proteins but play a key role in the regulation of gene expression and are involved in the formation of tumors. The expression of several microRNAs in various types of malignant tumors including liver cancer Or a decrease has been reported.

The tumor-derived exosome is a very small (50-100 nm) membrane structure that is produced in cancer cells and secreted into the serum. It contains several proteins inside, but it is known to contain tumor-derived microRNAs. The expression pattern of exosomal microRNA in the serum reflects microRNA expression in tumor cells, and a significant correlation has been reported between them (Taylor D et al., 2008). Exosomal microRNAs in the serum also show considerable stability, and exosomal microRNAs in serum are highly likely to be developed as new tumor biomarkers.

To date, studies on serum microRNAs as biomarkers for liver cancer have focused mostly on fewer patient samples and are still analyzing microRNAs that are detected in the blood. There are no liver cancer biomarkers using exosomal microRNAs secreted into the blood of liver cancer. Therefore, in order to discover useful serum microRNAs as liver cancer biomarkers, a systematic analysis study is required for large-scale serum samples of liver cancer patients and chronic liver disease patients whose clinical status at the time of obtaining the serum sample is accurately known.

Therefore, the present inventors have made efforts to develop a novel biomarker for diagnosis of liver cancer. As a result, the present inventors found that the expression pattern of specific microRNAs expressed in exosomes isolated from human blood (serum) was different in patients with liver cancer, and using this as a biomarker of novel liver cancer, The present invention has been completed.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, there is provided a method of detecting the presence or absence of one or more of Mir-101 (GenBank Accession Number: NR_029516), Mir-106b (GenBank Accession Number: NR_029831), Mir- (GenBank Accession Number: LM379069), Mir-342-3p (GenBank Accession Number: LM379258), Mir-16-5p (GenBank Accession Number: LM378756), Mir-26b-5p Wherein the exosomal miRNA comprises at least one exosomal miRNA selected from the group consisting of GenBank Accession Number LM378770 and Mir-22-3p (GenBank Accession Number: LM378764), and a biomarker composition for diagnosing liver cancer comprising the exosomal miRNA And an agent for measuring an expression level.

According to a preferred embodiment of the present invention, the composition is selected from the group consisting of Mir-18a (GenBank Accession Number: NR_029488), Mir-221 (GenBank Accession Number: LM608366), Mir- (GenBank Accession Number: LM608369). The expression level of at least one exosome miRNA may be measured by the method described above.

That is, the most important feature of the present invention is to diagnose and predict the onset of liver cancer using the exosomal miRNAs as a biomarker.

The term "miRNA (microRNA) " as used herein refers to approximately 22 nt of non-translated RNA that acts as a post-translational repressor through base linkage with the 3 'untranslated region (UTR) site of mRNA.

The biomarker of the present invention is excellent in the accuracy and reliability as a marker for liver cancer, and thus can be used for diagnosis of cancer development, development and / or metastasis.

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

In the present invention, the object to be detected is the corresponding exosomal miRNAs in the sample. That is, by detecting exosomal miRNAs, it is possible to confirm whether or not the above-mentioned liver cancer is caused.

The detection of exosomal miRNAs can usually be performed by extracting exosomes from the sample and detecting exosomal miRNAs in the extract. The detection of such exosome miRNAs can be measured by hybridization reaction and amplification reaction, but is not limited thereto and can be easily carried out using various techniques known in the art.

Further, according to a preferred embodiment of the present invention, the expression level of the exosome miRNA is measured from a biological sample of a patient suspected of having liver cancer.

The present invention is based on the finding that in the case of hepatocellular carcinoma, the serum levels of Mir-101, Mir-106b, Mir-122, Mir-195, Mir-150-5p, Mir-342-3p , Mir-106b, Mir-122, Mir-195, and Mir-16p based on confirming that the expression levels of Mir-16-5p, Mir-26b-5p and Mir- Mir-150-5p, Mir-342-3p, Mir-16-5p, Mir-26b-5p and Mir-22-3p are used as diagnostic markers for the incidence of liver cancer.

In addition, the present invention further provides a method for the treatment of liver cancer, wherein the level of expression of Mir-18a, Mir-221, Mir-222 and Mir-224 in blood is significantly increased compared to that of liver cancer (chronic hepatitis and / , Mir-18a, Mir-221, Mir-222 and Mir-224 are used as diagnostic markers for the onset of liver cancer.

The above-described chronic hepatitis and / or cirrhosis is known in the art as a comparative control against liver cancer. Since liver cancer occurs in patients with chronic hepatitis and / or liver cirrhosis, chronic hepatitis and / It should be used as a control group of patients.

That is, the biomarker of the present invention does not express or under-expresses a specific exosome miRNA; The expression level of the exosome miRNA in the exosome is examined in a subject having cancer cells or cancers in which specific exosome miRNAs are highly expressed.

The agent for measuring the expression level of the exosome miRNA of the present invention is an agent for measuring the expression level of the exosome miRNA, which is a marker whose expression level is decreased in a specimen of a biological specimen of a subject, Quot; means a molecule that can be used to detect a marker by identifying the level.

According to a preferred embodiment of the present invention, the agent for measuring the expression level of at least one of the above-mentioned exosome miRNAs is a primer or a probe that specifically binds to the exosome miRNAs.

That is, the detection of the nucleic acid can be performed by an amplification reaction using a nucleic acid molecule encoding the gene or one or more oligonucleotide primers hybridized to the nucleic acid molecule.

For example, the detection of exosome miRNAs using primers can be performed by amplifying the gene sequence using an amplification method such as PCR, and then confirming amplification of the gene by a method known in the art.

The primer is a nucleic acid sequence having a short free 3 'hydroxyl group, a short nucleic acid sequence capable of forming a base pair with a complementary template and serving as a starting point for template strand copying . Primers can be used to initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In the present invention, it is possible to diagnose the onset of liver cancer by performing PCR amplification using a sense and antisense primer that specifically binds to one or more of the above miRNAs and confirming the expression level thereof. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

A probe is labeled with a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or a long few tens of nucleotides. The probe may be prepared 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, hybridization is performed using a probe complementary to one or more of the miRNAs, and the level of expression can be confirmed, thereby diagnosing the incidence of liver cancer. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

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

For example, primers or probes can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", replacement of natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers, such as methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

Expression levels of miRNA can be measured according to methods commonly used in the field of biotechnology such as RT-PCR, competitive RT-PCR, real-time reverse transcriptase polymerase But are not limited to, real-time RT-PCR, RNase protection assay (RPA), northern blotting or gene chips.

According to another aspect of the present invention, there is provided a kit for the diagnosis of liver cancer comprising the composition.

The kit may include not only an agent for measuring the expression level of the exosome miRNA but also tools and reagents commonly used in the art that are suitably used as a diagnostic kit for liver cancer.

Examples of such tools or reagents include, but are not limited to, suitable carriers, markers capable of generating detectable signals, chromophores, solubilizers, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, Acrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose and combinations thereof.

For example, the diagnostic kit of the present invention may be a kit containing necessary elements necessary for performing RT-PCR. The RT-PCR kit can be used in combination with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase, in addition to the respective primer pairs specific for the marker miRNA Such as enzymes, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like.

Since the kit of the present invention includes the above-described biomarkers and compositions as constituent elements, redundant contents are omitted in order to avoid excessive complexity of the present invention.

According to still another aspect of the present invention, the present invention provides a method for providing information for diagnosis of liver cancer, comprising the steps of:

(a) separating the exosome from a biological sample of the subject; And

(b) The above exosomes are selected from the group consisting of Mir-101, Mir-106b, Mir-122, Mir-195, Mir-150-5p, Mir-342-3p, Mir- And comparing the expression level of at least one exosome miRNA selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 3 with the expression level of the corresponding exosomal miRNA of the comparative control sample.

In the present invention, (b) the above exosomes Mir-101, Mir-106b, Mir-122, Mir-195, Mir-150-5p, Mir- 342-3p, Mir- When the expression level of at least one exosome miRNA selected from the group consisting of Mir-22-3p is lower than that of the comparative control, the subject is judged to be liver cancer.

In addition, the composition may further include a step of measuring the expression level of at least one exosome miRNA selected from the group consisting of Mir-18a, Mir-221, Mir-222 and Mir-224 in the exosome.

When the expression level of at least one exosome miRNA selected from the group consisting of Mir-18a, Mir-221, Mir-222 and Mir-224 in the exosome is higher than that of the comparative control, the subject is judged to be liver cancer .

Unless otherwise specified herein, the term "control" as used herein refers to the level of exosome miRNA expression described above in a sample of a patient with liver cirrhosis or chronic hepatitis.

The prediction method of the present invention is characterized in that a biological sample is obtained from a subject and the expression level of the exosome miRNA is measured in the sample and then the expression of the above exosome miRNA is increased And determining that the sample is liver cancer cells or tissue when the expression is low (decreased).

The term "low expression" used herein to refer to the level of expression of the exosome miRNA as used herein refers to the level of expression of the exosome miRNA in a healthy, normal, or comparable subject when the biomarker exhibits or is indicative of an abnormal process, Refers to the value or level of a biomarker in a biological sample that is lower than the value or level range of the biomarker detected in the obtained biological sample.

The term "high expression" used herein to refer to the level of expression of the exosome miRNA as used herein refers to the level of expression of the exosome miRNA in a healthy, normal, or comparable subject when the biomarker exhibits or is indicative of an abnormal process, Refers to the value or level of a biomarker in a biological sample that is higher than the value or level range of the biomarker detected in the obtained biological sample.

Also, terms used in reference to the expression levels of the genes are referred to as " differential levels "or" differential values "or" differentially expressed " And includes both qualitative as well as quantitative differences in expression.

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

According to a preferred embodiment of the present invention, the biological sample is any one selected from the group consisting of blood, serum, saliva, biopsy, blood, skin tissue, liquid culture, feces and urine, But not limited to, serum, and can be prepared by treatment with a method commonly used in the technical field of the present invention.

Mir-101, Mir-106b, Mir-122, Mir-195, and Mir-150-b in the samples of patients suspected of developing or progressing liver cancer as compared to the expression level of the above- The expression level of at least one exosome miRNA selected from the group consisting of 5p, Mir-342-3p, Mir-16-5p, Mir-26b-5p and Mir-22-3p was significantly lowered, Can provide information.

In addition, when the expression level of one or more exosome miRNAs selected from the group consisting of Mir-18a, Mir-221, Mir-222 and Mir-224 is measured to be significantly higher, do.

The method of the present invention determines the onset of hepatocarcinoma using the biomarker described above, so redundant description is omitted in order to avoid excessive complexity of the present specification.

The microRNAs isolated from serum exosomes of patients with liver disease according to the present invention show specific expression patterns in liver cancer patients and can be used as liver cancer biomarkers. In the case of liver cancer, there is no serum biomarker with high sensitivity and specificity so far, and there is no marker to be used as a screening test for liver cancer. Exosomal microRNA biomarker secreted from serum of hepatocarcinoma cells developed through the present invention is a highly deadly liver cancer As a diagnostic method. In addition, there is no prognostic prediction biomarker that predicts the postoperative recurrence of patients with hepatocellular carcinoma or postoperative response to anticancer therapy such as sorafenib. Therefore, the biomarker developed in the present invention may be used as a predictor of hepatic cancer prognosis. It can be used for personalized medicine of liver cancer according to the decision of treatment method and prediction of prognosis.

Figure 1 shows the difference in serum exocase microRNA expression in patients with chronic hepatitis B (CHB), liver cirrhosis (LC), and liver cancer (HCC) by hepatitis B virus.
Figure 2 shows a serum exosomal microRNA array using a nanostructure array in patients with chronic hepatitis (CH) and liver cancer (HCC).

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example 1. Selection of exosomal miRNA (exosomal microRNA)

The present inventors isolated exosomal miRNAs from 20 sera from patients with chronic hepatitis B, cirrhosis and liver cancer caused by hepatitis B virus, and selected exosomal miRNAs that were increased or decreased in liver cancer.

First, the expression amounts of 10 total microRNAs (Mir-18a, Mir-21, Mir-93, Mir-101, Mir-106b, Mir-122, Mir-195, Mir-221, Mir- Were quantified using qPCR, and then compared in each chronic hepatitis patient, liver cirrhosis patient, and liver cancer patient group.

As a result, as shown in FIG. 1, the expression of Mir-18a, Mir-221, Mir-222 and Mir-224 in the serum exosomal microRNA was significantly increased in liver cancer patients compared with chronic hepatitis or cirrhosis patients <0.05). The expression of Mir-101, Mir-106b, Mir-122 and Mir-195 was significantly reduced in liver cancer patients compared to chronic hepatitis patients (P <0.05). Serum levels of Mir-21 and Mir-93 were not significantly different between the liver cancer patients and the chronic hepatitis patients (P = 0.925, P = 0.355) (Figure 1) (Sohn W, et al. : e184).

These results suggest that serum exosomal miRNAs can be used as blood biomarkers for liver cancer.

Example 2 Further Selection of Exosomal miRNA (exosomal microRNA)

Based on the results of Example 1, the present inventors have established a method for analyzing liver cancer exosome miRNA expression using a nanostring array (Nanostring microRNA) array, and compared the expression of serum exo gene miRNA in patients with hepatitis and liver cancer Respectively.

Although some studies have reported the use of chip-based arrays for free circulating microRNAs as a method for analyzing the expression pattern of serum exosomal miRNAs, it is difficult to accurately analyze the existing methods due to the low concentration of exosomal miRNAs. Meanwhile, Nanostring's nCounter miRNA expression assay instrument used in the present invention has an advantage that it can confirm the expression amount of about 700 types of microRNA genes known to date by one experiment. In particular, since the unnecessary gene amplification To reduce the masking effect, resulting in more accurate results.

After several experiments and consultation with the Nanostring head research team, we first established a method for analyzing serum exosomal miRNA expression profile of patients with chronic liver disease using Nanostring.

(Markers with a difference of more than 1.5-fold in serum exonome miRNA expression using nanostring arrays in patients with chronic hepatitis (CH) and liver cancer (HCC) are shown in red)

As a result, as shown in FIG. 2 and Table 1, serum exoose miRNA expression profiles of chronic hepatitis B (n = 5) and liver cancer (n = 6) Five different miRNAs (Mir-150-5p, Mir-342-3p, Mir-16-5p, Mir-26b-5p and Mir-22-3p) were significantly more potent in patients with hepatocellular carcinoma Expression was decreased.

Therefore, we suggest that these serum exosomal miRNAs can be used as serum biomarkers for new liver cancer.

In conclusion, the exosomal microRNA isolation technique and the first established Nanostring-based serum exosomal microRNA array analysis technique obtained in the embodiment of the present invention can be used for the diagnosis of liver cancer as well as biomarkers related to the prognosis It is expected to be useful for the research of liquid biopsy biomarkers of various malignant tumors in addition to liver cancer.

Claims (10)

(GenBank accession number: LM379069), Mir-342-3p (GenBank accession number: LM379258), Mir-16-5p (GenBank accession number: LM378756) ) And Mir-22-3p (GenBank Accession Number: LM378764) exosomal miRNA.
(GenBank accession number: LM379069), Mir-342-3p (GenBank accession number: LM379258), Mir-16-5p (GenBank accession number: LM378756) ) And Mir-22-3p (GenBank Accession Number: LM378764) exosomal miRNA.
The composition according to claim 2, wherein the composition is selected from the group consisting of Mir-101 (GenBank Accession Number: NR_029516), Mir-106b (GenBank Accession Number: NR_029831), Mir- 122 (GenBank Accession Number: NR_029667) (GenBank Accession Number: NR_029712), Mir-18a (GenBank Accession Number: NR_029488), Mir-221 (GenBank Accession Number: LM608366), Mir- Lt; RTI ID = 0.0 &gt; miRNA &lt; / RTI &gt; of the selected one or more selected.
3. The composition of claim 2, wherein the agent comprises a primer or a probe that specifically binds to the exosomal miRNA.
A kit for the diagnosis of liver cancer comprising the composition of any one of claims 2 to 4.
A method for providing information for diagnosis of liver cancer, comprising the steps of:
(a) separating the exosome from a biological sample of the subject; And
(b) the above-mentioned exosomes Mir-150-5p (GenBank Accession Number: LM379069), Mir-342-3p (GenBank Accession Number: LM379258), Mir- 16-5p (GenBank Accession Number: LM378756) 5p (GenBank Accession Number: LM378770) and Mir-22-3p (GenBank Accession Number: LM378764) exosomal miRNA with the expression level of the corresponding exosomal miRNA of the comparative control sample.
7. The method according to claim 6, wherein (b) the expression level of the exosomes Mir-150-5p, Mir-342-3p, Mir-16-5p, Mir-26b-5p and Mir- Wherein the subject is determined to be liver cancer when the expression is low compared to the control.
7. The method according to claim 6, wherein the method comprises the steps of: (a) obtaining a mutation in the exosomes of Mir-101 (GenBank Accession Number: NR_029516), Mir-106b (GenBank Accession Number: NR_029831), Mir- (GenBank Accession Number: NR_029712), Mir-18a (GenBank Accession Number: NR_029488), Mir-221 (GenBank Accession Number: LM608366) &Lt; / RTI &gt; wherein the method further comprises measuring the expression level of at least one exosome miRNA selected from the group consisting of: &lt; RTI ID = 0.0 &gt;
The method according to claim 8, wherein when the expression level of at least one exosome miRNA selected from the group consisting of Mir-18a, Mir-221, Mir-222 and Mir-224 in the exosome is higher than that of the control, Lt; RTI ID = 0.0 &gt; liver cancer. &Lt; / RTI &gt;
7. The method of claim 6, wherein the biological sample is any one selected from the group consisting of blood, serum, saliva, biopsy, skin tissue, liquid culture, feces and urine.

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