KR102108299B1 - MicroRNA Biomarker for Predicting Lymph Node Metastasis of Gastric Cancer - Google Patents

Abstract

The present invention relates to miRNA markers for predicting lymph node metastasis of gastric cancer, and more particularly, to predicting lymph node metastasis using miRNA expression profile in early gastric cancer in the mucosa. Prediction of lymph node metastasis using the expression of miRNA biomarker according to the present invention can be further improved by early application of gastric cancer by applying it before endoscopic submucosal exfoliation to patients with early gastric cancer, and the scope or method of gastric cancer surgery can be further improved. Because it can be clearly set, it is very useful for clinical applications.

Description

MicroRNA Marker for Predicting Lymph Node Metastasis of Gastric Cancer {MicroRNA Biomarker for Predicting Lymph Node Metastasis of Gastric Cancer}

The present invention relates to a microRNA (miRNA) marker for predicting lymph node metastasis of gastric cancer, and more particularly, to predicting lymph node metastasis using a miRNA expression profile in early gastric cancer in the mucosa.

Gastric cancer is the fourth most common cancer in the world, and the second highest cause of cancer death, especially in East Asian countries, including Korea. The incidence of gastric cancer ranks first in cancer in men and fourth in women.

In recent years, health checkups have become more common, and the incidence of early gastric cancer (EGC) is relatively faster in Korea than in advanced gastric cancer (AGC), so the proportion of early gastric cancer among all gastric cancers is 50. It is over%. Early gastric cancer, unlike advanced gastric cancer, has good treatment outcomes and has a 5-year survival rate of almost 90%. In the case of early gastric cancer, when the cancer is confined to the mucosa or only the surface layer of the submucosa (within ~ 500 μm) is infiltrated, endoscopic submucosal detachment according to its gross shape and histological type (endoscopic submucosal dissection, ESD). After receiving ESD, if the depth of cancer infiltration into submucosa is greater than 500 μm, or if cancer is observed in the blood vessels or lymphatic vessels, additional surgical resection of the stomach and lymph node extrusion of the surrounding lymph nodes are performed. In the case of confinement within, treatment with ESD was observed without additional treatment.

However, intramucosal gastric cancer (IMC) confined to the gastric mucosa may also metastasize to lymph nodes around the stomach. It has been reported that metastasis from about 1.9 to 6.4% of intramucosal gastric cancer to surrounding lymph nodes is found (Oh SY et al., Ann Surg . 265 (1): 137-142, 2017; Pyo JH et al., Ann Surg Oncol . 23 (Suppl 5): 784-791, 2016; Pyo JH et al., PLoS One. 11 (5): e0156207, 2016). These numbers may increase further if they are expanded to cancers that are targeted for ESD treatment. If the patient has only undergone ESD without knowing whether it has metastasized to the surrounding lymph nodes, the patient will endanger life with local recurrence and metastasis of the cancer. In other words, endoscopic submucosal dissection (ESD) has a high success rate in the treatment of gastric cancer, and is applied with open surgery to treat early gastric cancer, but diagnoses and predicts the metastasis of gastric cancer through endoscopic examination. I can't. If the gastric cancer tissue is excised only by endoscopic submucosal excision of the metastasized gastric cancer, the metastasized cancer is not completely cured, which presents a risk of recurrence. If it is possible to determine the metastasis in patients diagnosed with gastric cancer before surgery or removal of gastric cancer tissue such as endoscopic submucosal exfoliation, it is useful for selecting a tumor removal method and determining the surgical method and range before surgery, increasing the survival rate and surgery for gastric cancer patients. It will help reduce post-complications.

A study of gross classification or pathological indicators of tumors associated with lymph node metastasis in mucosal gastric cancer (Oh SY et al., Ann Surg . 265 (1): 137-142, 2017; Pyo JH et al., Ann Surg Oncol . 23 (Suppl 5): 784-791, 2016; Pyo JH et al., PLoS One. 11 (5): e0156207, 2016), but many predictions based on these pathological indicators have many variables, and the accuracy is not high by itself. In addition, it is known that the ring type cell carcinoma, a histological type with poor differentiation, has a lower incidence of lymph node metastasis or is similar to other types when confined within the mucosa (Hyung WJ et al., Cancer . 1; 94 (1): 78-83, 2002; Chiu CT et al., Dig Dis Sci . 56: 1749-56, 2011; Lee SH et al., Eur J Gastroenterol Hepatol . 27 (2): 170-4, 2015). There are limitations in predicting lymph node metastasis using only these previously known histopathological features. On the other hand, there are many studies to predict the prognosis or recurrence of gastric cancer patients through analysis of gene expression profiles, etc., but this is not only targeted for advanced gastric cancer, but it is difficult to apply the results clinically. However, there is no known reliable biomarker associated with lymph node metastasis in actual early gastric cancer.

As one of the new cancer and disease markers, miRNAs have been recently received, and miRNA research is underway for various cancers. However, miRNA as a predictor of lymph node metastasis in gastric cancer has not been identified.

Accordingly, the present inventors tried to find a method for efficiently predicting and confirming lymph node metastasis in patients with early gastric cancer, and as a result, intramucosal cancer (M-MGC) with lymph node metastasis among mucosal gastric cancer patients undergoing endoscopic submucosal dissection. ) To discover miRNA markers whose expression level changes significantly, and completed the present invention.

An object of the present invention is to find a miRNA marker capable of efficiently predicting lymph node metastasis in early gastric cancer, to measure the expression level of the miRNA marker, and to provide a method for providing information for predicting lymph node metastasis of gastric cancer compared to a control group.

Another object of the present invention is to provide a composition for predicting lymph node metastasis of gastric cancer comprising an agent capable of measuring the expression level of the miRNA and a kit for predicting lymph node metastasis of gastric cancer comprising the composition.

To achieve the above object, the present invention miR-3175, miR-4505, miR-3620-5p, miR-4459, miR-4507, miR-1587, miR-4720 from samples of patients with suspected lymph node metastasis of gastric cancer -5p, miR-4742-5p, miR-628-5p, miR-6779-5p, miR-106b-3p, miR-500a-3p, miR-502-3p, miR-574-3p, miR-1231, miR Measuring the expression level of any one or more miRNAs selected from the group consisting of -151b, miR-6831-5p, miR-125a-5p, miR-486-5p, miR-181d-5p and miR-3609; And comparing the measured expression level of one or more miRNAs with a normal control sample.

The present invention also miR-3175, miR-4505, miR-3620-5p, miR-4459, miR-4507, miR-1587, miR-4720-5p, miR-4742-5p, miR-628-5p, miR -6779-5p, miR-106b-3p, miR-500a-3p, miR-502-3p, miR-574-3p, miR-1231, miR-151b, miR-6831-5p, miR-125a-5p, miR Provided is a composition for predicting lymph node metastasis of gastric cancer, comprising an agent capable of measuring the expression level of any one or more miRNAs selected from the group consisting of -486-5p, miR-181d-5p, and miR-3609.

The present invention also provides a kit for predicting lymph node metastasis of gastric cancer comprising the composition.

Prediction of lymph node metastasis using the expression of miRNA biomarker according to the present invention can be further improved by early application of gastric cancer by applying it before endoscopic submucosal exfoliation to patients with early gastric cancer, and the scope or method of gastric cancer surgery can be further improved. Because it can be clearly set, it is very useful for clinical applications.

1 is a schematic diagram of miRNA Affymetrix GeneChip miRNA 4.0 Array for finding miRNAs that significantly increase or decrease only in intramucosal cancer with lymph node metastasis (M-MGC).
2 shows 11 miRNAs with increased expression in mucosal gastric cancer (M-IMC) with lymph node metastasis.
3 shows 10 miRNAs with reduced expression in mucosal gastric cancer (M-IMC) with lymph node metastasis.
4 shows 10 miRNAs having known functions among 21 miRNAs.
5 is a result of predicting lymph node metastasis of 21 miRNAs using a leave-one-out-cross-validation (LOOCV) method, and analyzing the results of ROC (receiver operating characteristic).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known in the art and is commonly used.

The present invention relates to a method for detecting lymph node metastasis markers of early gastric cancer and effectively predicting lymph node metastasis of gastric cancer by using a nucleic acid specifically binding to the marker. The method using the miRNA marker of the present invention enables early predictability of lymph node metastasis of gastric cancer, thus leading to early treatment and improvement in prognosis, and also to monitor the effectiveness of surgical, radiotherapy, and chemotherapy treatments. It makes possible.

In the present invention, 21 kinds of miRNAs whose expression levels are significantly changed in normal tissues, intramucosal gastric cancer without lymph node metastasis (N-IMC) and intramucosal gastric cancer with lymph node metastasis (M-IMC) are selected, and their expression profiles Through this, it was confirmed that the lymph node metastasis of early gastric cancer can be effectively predicted.

Therefore, the present invention in one aspect (a) miR-3175, miR-4505, miR-3620-5p, miR-4459, miR-4507, miR-1587, miR- from samples of patients with suspected lymph node metastasis of gastric cancer 4720-5p, miR-4742-5p, miR-628-5p, miR-6779-5p, miR-106b-3p, miR-500a-3p, miR-502-3p, miR-574-3p, miR-1231, measuring the expression level of any one or more miRNAs selected from the group consisting of miR-151b, miR-6831-5p, miR-125a-5p, miR-486-5p, miR-181d-5p and miR-3609; And (b) comparing the measured expression level of one or more miRNAs with a normal control sample.

In the present invention, compared to the control group miR-3175, miR-4505, miR-3620-5p, miR-4459, miR-4507, miR-1587, miR-4720-5p, miR-4742-5p, miR-628- When the expression level of any one or more miRNAs selected from the group consisting of 5p and miR-6779-5p increases, or, compared to the control group, miR-106b-3p, miR-500a-3p, miR-502-3p, miR- 574-3p, miR-1231, miR-151b, miR-6831-5p, miR-125a-5p, miR-486-5p, miR-181d-5p and any one or more miRNA selected from the group consisting of miR-3609 When the expression level decreases, it is preferable to determine that there is a risk of lymph node metastasis of gastric cancer, but is not limited thereto.

As used herein, the terms “miRNA” or “microRNA” or “miR” are those whose precursor RNA transcript can form a small stem-loop from which the mature “miRNA” is an endonuclease die Refers to an RNA (or RNA analog) containing the product of an endogenous, non-encoded gene that is cleaved by a Dicer. miRNAs are encoded in genes that are distinct from the mRNAs that regulate their expression.

In one example, the term “miRNA” or “microRNA” refers to a single-stranded RNA molecule of at least 10 nucleotides and up to 35 nucleotides covalently linked together. The miRNA or their complementary strand molecules include, but are not limited to, 15 to 50, preferably 15 to 30, and more preferably 18 to 25 nucleic acids.

The sequence of miRNA as described herein includes the miRNA of SEQ ID NO: 1 to SEQ ID NO: 21 in Table 1 below.

Sequence number miRNA miRBase Accession No. One miR-3175 MIMAT0015052 2 miR-4505 MIMAT0019041 3 miR-3620-5p MIMAT0022967 4 miR-4459 MIMAT0018981 5 miR-4507 MIMAT0019044 6 miR-1587 MIMAT0019077 7 miR-4720-5p MIMAT0019833 8 miR-4742-5p MIMAT0019872 9 miR-628-5p MIMAT0004809 10 miR-6779-5p MIMAT0027458 11 miR-106b-3p MIMAT0004672 12 miR-500a-3p MIMAT0002871 13 miR-502-3p MIMAT0004775 14 miR-574-3p MIMAT0003239 15 miR-1231 MIMAT0005586 16 miR-151b MIMAT0010214 17 miR-6831-5p MIMAT0027562 18 miR-125a-5p MIMAT0000443 19 miR-486-5p MIMAT0002177 20 miR-181d-5p MIMAT0002821 21 miR-3609 MIMAT0017986

As can be appreciated by those skilled in the art, miRNA is a type of polynucleotide having a sequence comprising a letter such as "ATGC". The nucleotides are 5 '→ 3' in order from left to right, unless otherwise indicated "A" is deoxyadenoside, "C" is deoxycytidine, "G" is doxyguanoside, and "T" is deoxy. Thymidine. Letters A, C, G, and T are standard in the art and can be used to refer to the base itself, a nucleoside, or a nucleotide comprising a base. The term is an oligonucleotide consisting of naturally occurring nucleobases, sugars, and internucleotide (skeletal) linkages, and also oligonucleotides having non-naturally occurring sites with similarly or specifically improved action. It includes. In naturally occurring polynucleotides, the internucleoside linkage is typically a phosphodiester bond, and the subunit is referred to as a "nucleotide". The term "oligonucleotide" can also include fully or partially modified or substituted oligonucleotides such as bases and / or sugars.

As can be appreciated in the art of the present disclosure, in any of the methods described herein, the miRNA may not have 100% sequence identity with the sequence of the miRNAs listed in Table 1 above.

In the present invention, the step of measuring the expression level of the one or more miRNA may 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 polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting or gene chip, etc. It does not work.

In the present invention, the sample of a patient with suspected lymph node metastasis of gastric cancer is preferably, but is not limited to, tissue or cells isolated from the mucosa or submucosa of the stomach.

In the present invention, the gastric cancer of the present invention is preferably an early gastric cancer (EGC), more preferably an intramucosal gastric cancer (IMC), but is not limited thereto.

As used herein, the term “cancer” is representative of cancer-causing cells, such as unregulated proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain characteristic morphological properties known in the art. It refers to the presence of characteristic cells. In one example, the “cancer” can be gastric cancer or gastrointestinal cancer. In one example, “cancer” can include not only malignant cancer, but also pre-malignant cancer. Accordingly, the term "stomach cancer" includes all stages of gastric cancer as described by the National Cancer Institute of the National Institutes of Health.

As used herein, in any of the methods described herein, the terms “subject” and “patient” are interchanged to refer to animals (eg, mammals, fish, amphibians, reptiles, birds and insects). Can be used. In one example, the subject can be a mammal (eg, a non-human mammal (eg, dog, cat, or primate) and human). In one example, the subject can be a primate (eg, chimpanzee and human). In one example, the subject can be a human. In one example, the subject may be a person with or without gastric cancer (or gastrointestinal cancer).

In another aspect, the present invention, miR-3175, miR-4505, miR-3620-5p, miR-4459, miR-4507, miR-1587, miR-4720-5p, miR-4742-5p, miR-628-5p , miR-6779-5p, miR-106b-3p, miR-500a-3p, miR-502-3p, miR-574-3p, miR-1231, miR-151b, miR-6831-5p, miR-125a-5p , miR-486-5p, miR-181d-5p and relates to a composition for predicting lymph node metastasis of gastric cancer comprising an agent capable of measuring the expression level of any one or more miRNAs selected from the group consisting of miR-3609.

In another aspect of the present invention, an agent capable of measuring the expression level of the miRNA may be characterized by including a probe specifically binding to the miRNA of Table 1 or a primer capable of amplifying the miRNA. .

The primer is a short nucleic acid sequence having a short free 3 'hydroxyl group, which can form a complementary template and base pair, and serves as a starting point for template strand copying. Means sequence. Primers can be synthesized DNA in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures and four different nucleoside triphosphates. In the present invention, PCR amplification is performed using sense and antisense primers that specifically bind to one or more miRNAs of the stomach to confirm expression level, thereby diagnosing metastasis of gastric cancer to lymph nodes. 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 a few bases to tens of bases in short, and is labeled. 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. In the present invention, it is possible to diagnose whether metastasis of gastric cancer to a lymph node is performed by confirming the expression level by performing hybridization using a probe complementary to one or more miRNAs of the stomach. The appropriate probe selection and hybridization conditions can be modified based on those known in the art.

These primers or probes can be appropriately designed by those skilled in the art based on the sequence of a known miRNA.

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, "capping", substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (eg methyl phosphonate, phosphotriester, Phosphoroamidates, carbamates, etc.) or modifications to charged linkages (eg, phosphorothioate, phosphorodithioate, etc.).

In another aspect, the present invention, miR-3175, miR-4505, miR-3620-5p, miR-4459, miR-4507, miR-1587, miR-4720-5p, miR-4742-5p, miR-628- 5p, miR-6779-5p, miR-106b-3p, miR-500a-3p, miR-502-3p, miR-574-3p, miR-1231, miR-151b, miR-6831-5p, miR-125a- Gastric cancer comprising a composition for predicting lymph node metastasis of gastric cancer comprising an agent capable of measuring the expression level of any one or more miRNAs selected from the group consisting of 5p, miR-486-5p, miR-181d-5p and miR-3609 It relates to a kit for predicting lymph node metastasis.

The composition for diagnosing lymph node metastasis of gastric cancer of the present invention may include other components for making it suitable for use as a kit for predicting lymph node metastasis of gastric cancer in addition to an agent for measuring the expression level of one or more miRNAs in the stomach. That is, in addition to the composition, it may be to further include a configuration commonly used in the prediction kit.

For example, the prediction 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 capable of amplifying the marker miRNA. Enzymes such as, DNase, RNAse inhibitors, DEPC-water (DEPC-water), and may include sterile water.

The prediction kit of the present invention may be a gene chip kit. The gene chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, agents, enzymes, and the like for preparing a fluorescent marker probe. Further, the substrate may include cDNA corresponding to a quantitative control gene or a fragment thereof.

The kit can also include nucleic acid template (s) for positive and / or negative control reactions. The kit can also include any reaction components or buffers necessary for the isolation of miRNAs from samples obtained from subjects. In addition, a fluorescent material may be additionally included in the kit, and the fluorescent material is composed of a strepavidin-alkaline phosphatase conjugate, a chemiflurorensce, and a chemiluminescent material. It is preferably selected from the group, but is not limited thereto.

The invention illustratively described herein may be suitably practiced in the absence of any component or ingredients, limitations or limitations not specifically disclosed herein. Thus, for example, the terms "comprising", "comprising", "containing", etc., will be read broadly and without limitation. Also, the terms and expressions used herein are used as and without limitation in the description, and there is no intention in the use of these terms and expressions except for any equivalents of the indicated or described features or parts thereof, but various modifications are patented. It is recognized that it is possible within the claimed area. Thus, while the present invention has been specifically disclosed by preferred embodiments and any features, the modifications and variations of the invention as embodied herein disclosed may be reclassified by those skilled in the art, and such modifications and variations of the present invention It is considered to be within the domain.

[ Example ]

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Stomach cancer patients Experimental group

Samples from the experimental group were selected as 16 gastric cancer tissues whose metastasis to the surrounding lymph nodes was confirmed among stage Ta 1a (a tumor is localized on the mucous membrane) among patients undergoing gastric cancer surgery at Ajou University Hospital from 2006 to 2012. The control group was selected from 12 gastric cancer tissues of patients who had gastric cancer at Ajou University Hospital from 2012 to 2013 with stage Ta 1a and no lymph node metastasis, and did not relapse during follow-up period of 3 years or more. Among these patients, normal gastric tissue was selected from 7 gastric tissues, and the study was conducted as another control group.

This study was conducted in accordance with the Ethics Code of the World Medical Association (Declaration of Helsinki) and was approved by the Institutional Review Board (IRB) of Ajou University Hospital.

Example  One: Affymetrix GeneChip miRNA  4.0 Array

Total RNA from sample tissue was extracted using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. RNA concentration was quantified using a nanodrop (NanoDrop, USA), and RNA purity was measured using a 2100 bioanalyzer (Agilent Technologies, CA, USA). The extracted RNA was stored at -80 ° C prior to microarray analysis. miRNA microarray analysis was performed within 2 days of RNA extraction.

Microarrays were performed on a total of 35 RNA samples, and the Affymetrix Genechip miRNA 4.0 array experiment followed the manufacturer's protocol.

35 RNA samples of 130 ng each were labeled using the FlashTag ™ Biotin RNA Labeling Kit (Genisphere, Hatfield, PA, USA), and then placed at 99 ° C for 5 minutes and 45 ° C for 5 minutes. RNA-array hybridization was performed for 16 hours on an Affymetrix® 450 Fluidics Station instrument. The hybridized chip was washed with Genechip Fluidics Station 450 (Affymetrix, Santa Clara, California, United States), and then scanned using an Affymetrix GCS 3000 canner (Affymetrix, Santa Clara, California, United States). After the scan was completed, chip QC and RNA normalization were performed using Affymetrix 'GeneChip ™ Expression Console software.

85 genes were selected by comparing the expression between normal tissue and intramucosal cancer with lymph node metastasis and comparing the expression between intramucosal cancer without lymph node metastasis and intramucosal cancer with lymph node metastasis. Then, 27 genes overlapped in each group were selected, and 21 miRNAs except 6 snoRNAs (small nucleolar RNAs) were identified (FIG. 1). The miRNAs were selected only by miRNAs having a difference from normal, and were selected with focus on function.

The expression of the excavated 21 miRNAs was relatively compared in normal tissue, intramucosal gastric cancer without lymph node metastasis (N-IMC) and intramucosal gastric cancer with lymph node metastasis (M-IMC). As a result, 10 types of miRNA miR-3175, miR-4505, miR-3620-5p, miR-4459, miR-4507, miR-1587, miR-4720-5p, miR-4742-5p, miR-628-5p And miR-6779-5p increased significantly in M-IMC only (FIG. 2), 11 miRNA miR-106b-3p, miR-500a-3p, miR-502-3p, miR-574-3p, miR-1231, miR-151b, miR-6831-5p, miR-125a-5p, miR-486-5p, miR-181d-5p and miR-3609 significantly decreased expression in M-IMC only (FIG. 3) .

Example  2: miRNA  Functional analysis of target genes

To demonstrate the reliability and usefulness of 21 miRNAs discovered in Example 1, functional pathways of 10 miRNAs with known functions were analyzed (Table 2). Table 2 is an analysis using an online database called Tarbase (DIANA tool), and is a program that informs the signaling pathways associated with the miRNAs based on the experimentally proven miRNA target genes.

As a result, 10 types of miRNA miR-3175, miR-628-5p, miR-106b-3p, miR-500a-3p, miR-502-3p, miR-574-3p, miR-1231, miR-151b, miR As shown in the table above, -125a-5p and miR-486-5p were found to play an important role in the development and progression of cancer, including colorectal cancer, pathways in cancer, cell cycle, neutrophin signaling pathway, and transcriptional misregulation in cancer. . In particular, it was confirmed that it is related to adherens junction, HIF-1 signaling and focal adhesion, which are key pathways for cancer metastasis as an angiogenic signaling pathway known to be associated with tumor metastasis (FIG. 4).

That is, the target genes inhibited by the miRNA of the present invention have been shown to regulate the functions involved in tumor angiogenesis and tumor metastasis promotion.

Example  3: Multiple miRNA Marker  Evaluation of predictive performance of lymph node metastasis in gastric cancer by combination

BRB-array tool software (Version 2.13.2 for × 64 systems, NCI, Bethesda, MD) to predict lymph node metastasis using 21 miRNAs dysregulated in intramucosal gastric cancer (M-IMC) with lymph node metastasis ) Was used. BRB-array tool provides CCP, DLDA, and BCCP algorithms that execute leave one out cross validation (LOOCV), a method of validation.

Therefore, as a result of analyzing the receiver-operating characteristic (ROC) using the LOOCV method, AUC = 0.917 (CCP), 0.911 (DLDA), and 0.88 (BCCP) were shown (FIG. 5). That is, it was confirmed that the combination of 21 miRNAs in Table 1 can effectively predict lymph node metastasis.

As the specific parts of the present invention have been described in detail above, it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> MicroRNA Biomarker for Predicting Lymph Node Metastasis of          Gastric Cancer <130> P17-B209 <160> 21 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3175 <400> 1 cggggagaga acgcagugac gu 22 <210> 2 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-4505 <400> 2 aggcugggcu gggacgga 18 <210> 3 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3620-5p <400> 3 gugggcuggg cugggcuggg cc 22 <210> 4 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-4459 <400> 4 ccaggaggcg gaggaggugg ag 22 <210> 5 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-4507 <400> 5 cuggguuggg cugggcuggg 20 <210> 6 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1587 <400> 6 uugggcuggg cuggguuggg 20 <210> 7 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-4720-5p <400> 7 ccuggcauau uugguauaac uu 22 <210> 8 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-4742-5p <400> 8 ucaggcaaag ggauauuuac aga 23 <210> 9 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-628-5p <400> 9 augcugacau auuuacuaga gg 22 <210> 10 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-6779-5p <400> 10 cugggagggg cuggguuugg c 21 <210> 11 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-106b-3p <400> 11 ccgcacugug gguacuugcu gc 22 <210> 12 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-500a-3p <400> 12 augcaccugg gcaaggauuc ug 22 <210> 13 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-502-3p <400> 13 aaugcaccug ggcaaggauu ca 22 <210> 14 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-574-3p <400> 14 cacgcucaug cacacaccca ca 22 <210> 15 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1231 <400> 15 gugucugggc ggacagcugc 20 <210> 16 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-151b <400> 16 ucgaggagcu cacagucu 18 <210> 17 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-6831-5p <400> 17 uagguagagu gugaggagga gguc 24 <210> 18 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-125a-5p <400> 18 ucccugagac ccuuuaaccu guga 24 <210> 19 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-486-5p <400> 19 uccuguacug agcugccccg ag 22 <210> 20 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-181d-5p <400> 20 aacauucauu guugucggug ggu 23 <210> 21 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3609 <400> 21 caaagugaug aguaauacug gcug 24

Claims (15)

Informational methods for predicting lymph node metastasis in gastric cancer, including the following steps:
(a) miR-3175, miR-4505, miR-3620-5p, miR-4459, miR-4507, miR-1587, miR-4720-5p, miR-4742- from samples of patients with suspected lymph node metastasis of gastric cancer 5p, miR-628-5p, miR-6779-5p, miR-106b-3p, miR-500a-3p, miR-502-3p, miR-574-3p, miR-1231, miR-151b, miR-6831- Measuring the expression levels of miRNAs of 5p, miR-125a-5p, miR-486-5p, miR-181d-5p and miR-3609; And
(B) comparing the expression level of the measured miRNA with a normal control sample.
According to claim 1, wherein the step (b) is miR-3175, miR-4505, miR-3620-5p, miR-4459, miR-4507, miR-1587, miR-4720-5p, miR-4742 compared to the control group Lymph node metastasis of gastric cancer characterized by determining that there is a risk of lymph node metastasis of gastric cancer when the expression level of any one or more miRNAs selected from the group consisting of -5p, miR-628-5p and miR-6779-5p is increased Method of providing information for prediction.
The method of claim 1, wherein the step (b) is miR-106b-3p, miR-500a-3p, miR-502-3p, miR-574-3p, miR-1231, miR-151b, miR-6831 compared to the control group. When the expression level of any one or more miRNAs selected from the group consisting of -5p, miR-125a-5p, miR-486-5p, miR-181d-5p, and miR-3609 decreases, it is said that there is a risk of lymph node metastasis of gastric cancer Method for providing information for predicting lymph node metastasis of gastric cancer, characterized by determining.
According to claim 1, The step of measuring the expression level of the miRNA is measured by reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or gene chip Method for providing information for predicting lymph node metastasis of gastric cancer, characterized in that.
The method of claim 1, wherein the sample is tissue or cells separated from the mucosa or submucosa of the stomach.
The method of claim 1, wherein the gastric cancer is early gastric cancer (EGC).
The method of claim 6, wherein the gastric cancer is intramucosal gastric cancer (IMC).
miR-3175, miR-4505, miR-3620-5p, miR-4459, miR-4507, miR-1587, miR-4720-5p, miR-4742-5p, miR-628-5p, miR-6779-5p, miR-106b-3p, miR-500a-3p, miR-502-3p, miR-574-3p, miR-1231, miR-151b, miR-6831-5p, miR-125a-5p, miR-486-5p, A composition for predicting lymph node metastasis of gastric cancer, comprising an agent capable of measuring the expression level of miRNA of miR-181d-5p and miR-3609.
The composition for predicting lymph node metastasis of gastric cancer according to claim 8, comprising a probe specifically binding to the miRNA or a primer capable of amplifying the miRNA.
The composition for predicting lymph node metastasis of gastric cancer according to claim 8, wherein the gastric cancer is early gastric cancer (EGC).
The composition of claim 10, wherein the gastric cancer is intramucosal gastric cancer (IMC).
A kit for predicting lymph node metastasis of gastric cancer comprising the composition of claim 8 or 9.
The kit for predicting lymph node metastasis of gastric cancer according to claim 12, wherein the kit is an RT-PCR kit or a gene chip kit.
The kit for predicting lymph node metastasis of gastric cancer according to claim 12, wherein the gastric cancer is early gastric cancer (EGC).
The kit for predicting lymph node metastasis of gastric cancer according to claim 14, wherein the gastric cancer is intramucosal gastric cancer (IMC).

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