WO2022075774A1 - Biomarker for predicting metastasis of pancreatic cancer and use thereof - Google Patents

Biomarker for predicting metastasis of pancreatic cancer and use thereof Download PDF

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WO2022075774A1
WO2022075774A1 PCT/KR2021/013798 KR2021013798W WO2022075774A1 WO 2022075774 A1 WO2022075774 A1 WO 2022075774A1 KR 2021013798 W KR2021013798 W KR 2021013798W WO 2022075774 A1 WO2022075774 A1 WO 2022075774A1
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pancreatic cancer
protein
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metastasis
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윤승배
송미영
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가톨릭대학교 산학협력단
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  • the present invention provides a gene comprising SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) It provides a marker composition for predicting metastasis of pancreatic cancer, comprising the mRNA or protein encoded by the gene.
  • the present invention provides mRNA of a gene comprising SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) or the gene is encoded by the gene It provides a composition for predicting metastasis of pancreatic cancer, including an agent for measuring the protein level, and a kit for predicting metastasis of pancreatic cancer comprising the same.
  • SERPINB12 Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2
  • MMRN1 Multimerin-1; NM_001371403.1, NM_007351.3
  • the present inventors analyzed the protein in the exosomes isolated from the plasma of normal persons and pancreatic cancer patients, and discovered a gene whose expression was significantly increased in pancreatic cancer patients showing liver metastasis compared to early pancreatic cancer patients. It is expected that it will be usefully used as a biomarker for predicting potential.
  • NM_007351.3 is an analysis of the protein in the exosomes isolated from plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly in pancreatic cancer patients showing liver metastasis MMRN1 (Multimerin-1; NCBI accession number: NM_001371403) .1, the result showing that the expression level of NM_007351.3) increases.
  • the present invention provides mRNA of a gene comprising SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) or the gene is encoded by the gene It provides a marker composition for predicting metastasis of pancreatic cancer, including a protein.
  • pancreatic cancer refers to an aggressive characteristic in which pancreatic cells divide and grow ignoring normal growth limits, an invasive characteristic that penetrates into surrounding tissues, and a metastatic (metastatic) characteristic that spreads to other parts of the body.
  • Metastatic refers to diseases caused by cells with characteristics.
  • the expression of specific genes (SERPINB12, MMRN1, HPSE, AZGP1, ACTN2, APP, ARG1, MMP9, LACRT, KRT3 and GSDMA) in a sample of a pancreatic cancer patient showing liver metastasis compared to an early pancreatic cancer patient is significant It was confirmed that the increase was significantly increased (see Example 2).
  • the kit of the present invention may be a kit including essential elements necessary for performing RT-PCR.
  • the RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC -Water (DEPC-water), sterile water, etc. may be included.
  • dNTPs deoxynucleotides
  • enzymes such as Taq-polymerase and reverse transcriptase
  • DNase DNase
  • RNase inhibitors DEPC -Water
  • sterile water etc.
  • a primer pair specific for a gene used as a quantitative control may be included.
  • the present invention provides SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) in a biological sample derived from a subject. ) It provides an information providing method for predicting metastasis of pancreatic cancer, comprising measuring the level of the mRNA of the gene or the protein encoded from the gene.
  • the mRNA level is determined by next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase protection assay; RPA), microarray (microarray), and may be measured through one or more methods selected from the group consisting of northern blotting (northern blotting), but is not limited thereto.
  • NGS next generation sequencing
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • Real-time PCR real-time polymerase chain reaction
  • RPA RNase protection assay
  • microarray microarray
  • Plasma samples from normal and pancreatic cancer patients are collected in BD Vacutainer Plastic K2EDTA Tube (BD, Cat.no 367525), and plasma is separated by centrifugation within 2 hours at 2,500 rpm and 4°C for 20 minutes. The supernatant plasma was aliquoted into Protein LoBind Tubes (Eppendorf, Cat.no 0030108116) by 500 ⁇ l and stored at -80°C.
  • Plasma samples were centrifuged at 3,000 g and 4 °C for 15 minutes to remove cells and cell debris in the plasma. Then, 250 ⁇ l of Exo2DTM for Protein assay (EXOSOMEplus) was added to 500 ⁇ l of plasma sample at a ratio of 2: 1 to isolate exosomes based on the Aqueous Two-Phase System, HaltTM Protease and Phosphatase Inhibitor Cocktail, EDTA-Free (Thermo scientific) was added to 200 ⁇ l of exosomes in PBS to prepare exosome samples by resuspending them. Samples were stored at -80 °C until analysis.
  • EXOSOMEplus Exo2DTM for Protein assay

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Abstract

The present invention relates to a biomarker for predicting the metastasis of pancreatic cancer and use thereof and, more particularly, to a marker composition for predicting the metastasis of pancreatic cancer, a composition for predicting the metastasis of pancreatic cancer, a prediction kit, and a method for providing information for predicting the metastasis of pancreatic cancer. Through analysis of a protein in exosomes isolated from plasma of normal individuals and pancreatic cancer patients, the inventors of the present invention discovered a gene of which the expression was significantly increased in pancreatic cancer patients showing liver metastasis compared to early pancreatic cancer patients. Thus, the gene is expected to be effectively used as a biomarker for predicting the metastatic potential of pancreatic cancer.

Description

췌장암의 전이를 예측하기 위한 바이오마커 및 이의 용도Biomarkers for predicting metastasis of pancreatic cancer and uses thereof
본 발명은 췌장암의 전이를 예측하기 위한 바이오마커 및 이의 용도에 관한 것으로, 보다 구체적으로는 췌장암의 전이 예측용 마커 조성물, 췌장암 전이 예측용 조성물, 예측용 키트, 및 췌장암의 전이 예측을 위한 정보제공방법에 관한 것이다.The present invention relates to a biomarker for predicting metastasis of pancreatic cancer and its use, and more particularly, to a marker composition for predicting metastasis of pancreatic cancer, a composition for predicting pancreatic cancer metastasis, a kit for prediction, and information for predicting metastasis of pancreatic cancer it's about how
췌장암은 췌장에서 기원한 악성 종양으로, 대부분의 환자가 암이 진행된 상태로 발견되기 때문에 5년 생존율이 10%도 되지 않는 암으로 알려져있다. 췌장암의 국내 발생률은 8위에 이르지만, 암에 의한 사망에는 폐암, 간암, 위암, 대장암 바로 다음을 차지하고 있다. 췌장암의 증상으로는 여러 가지 췌장 질환에서 볼 수 있는 증상이 나타날 수 있으며, 복통, 식욕부진, 체중감소, 황달 등이 가장 흔한 증상으로 췌장암 환자의 대부분에서 복통과 체중감소가 나타나고, 췌두부암 환자의 대부분에서 황달이 나타난다.Pancreatic cancer is a malignant tumor originating from the pancreas, and it is known that the 5-year survival rate is less than 10% because most patients are found to have advanced cancer. Although the incidence of pancreatic cancer in Korea ranks eighth, cancer-related deaths account for just after lung cancer, liver cancer, stomach cancer, and colorectal cancer. Symptoms of pancreatic cancer include symptoms found in various pancreatic diseases, and abdominal pain, anorexia, weight loss, and jaundice are the most common symptoms. Jaundice occurs in most cases.
한편, 암전이는 다양한 유전적 또는 후생유전적인 변이가 결집된 결과로 발생하는 매우 복잡한 과정이며, 특정 유전자의 발현 증가 혹은 감소가 다양한 세포 내 신호전달체계가 단독 또는 서로 영향을 받으며 전이의 각 단계를 조절할 것으로 유추된다.On the other hand, cancer metastasis is a very complex process that occurs as a result of the aggregation of various genetic or epigenetic mutations, and the increase or decrease in the expression of a specific gene is influenced by various intracellular signaling systems alone or each other, and each stage of metastasis It is presumed to control
따라서 특정 단백질의 발현에 의한 전이는 그 유전자의 발현 증가 또는 감소에 의한 직접적인 결과로 이해하기 보다는 그 유전자의 발현에 의해 조절되는 신호전달 경로의 변화에 기인한 결과로 이해되어야 할 것이다.Therefore, metastasis due to the expression of a specific protein should be understood as a result due to a change in the signaling pathway regulated by the expression of the gene rather than a direct result by the increase or decrease in the expression of the gene.
대한민국 등록특허 제1925125호는 개체의 혈액 시료에서 NCKAP1 단백질의 발현량을 검출하여 대장암 전이를 예측하는 방법을 개시하고 있으며, 대한민국 등록특허 제1895423호는 전립선암의 암전이를 예측하기 위한 바이오마커로서 CCM1 단백질의 발현을 억제하기 위한 shRNA 또는 siRNA를 포함하는 전립선암 치료용 조성물을, 대한민국 공개특허 제10-2020-0095159호 에서는 폐암 전이 예측용 바이오마커 조성물을 개시하고 있다. 하지만, 췌장암의 전이를 예측하기 위한 효과적인 바이오마커에 대한 연구는 미미한 실정이다.Korean Patent No. 1925125 discloses a method for predicting colorectal cancer metastasis by detecting the expression level of NCKAP1 protein in a blood sample of an individual, and Korean Patent No. 1895423 discloses a biomarker for predicting cancer metastasis of prostate cancer. A composition for treating prostate cancer including shRNA or siRNA for inhibiting the expression of CCM1 protein, and Korean Patent Laid-Open No. 10-2020-0095159 discloses a biomarker composition for predicting lung cancer metastasis. However, studies on effective biomarkers for predicting pancreatic cancer metastasis are insignificant.
상기와 같은 문제점을 해결하기 위하여, 본 발명자들은 췌장암의 전이를 예측할 수 있는 바이오마커를 개발하고자 초기 췌장암 환자 및 간 전이를 나타낸 췌장암 환자에서 혈장을 채취하고 이로부터 엑소좀(exosome)을 분리하여 엑소좀 단백질을 분석한 결과, 간 전이를 나타내는 췌장암 환자에서만 유의하게 발현이 증가하는 유전자를 발견하였는 바, 이에 기초하여 본 발명을 완성하였다.In order to solve the above problems, the present inventors collected plasma from patients with early pancreatic cancer and pancreatic cancer patients showing liver metastasis in order to develop a biomarker that can predict metastasis of pancreatic cancer, and separated exosomes from the exosomes. As a result of analyzing some proteins, it was found that a gene whose expression was significantly increased only in pancreatic cancer patients with liver metastasis was found. Based on this, the present invention was completed.
이에 본 발명은 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3)을 포함하는 유전자의 mRNA 또는 상기 유전자가 암호화하는 단백질을 포함하는, 췌장암의 전이 예측용 마커 조성물을 제공한다.Accordingly, the present invention provides mRNA of a gene including SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) or the gene encoded by the gene It provides a marker composition for predicting metastasis of pancreatic cancer, including a protein.
또한, 본 발명은 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3)을 포함하는 유전자의 mRNA 또는 상기 유전자가 암호화하는 단백질 수준을 측정하는 제제를 포함하는, 췌장암의 전이 예측용 조성물 및 상기 조성물을 포함하는 췌장암의 전이 예측용 키트를 제공한다.In addition, the present invention provides mRNA of a gene comprising SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) or the gene is encoded by the gene It provides a composition for predicting metastasis of pancreatic cancer, and a kit for predicting metastasis of pancreatic cancer comprising the composition, including an agent for measuring the protein level.
또한, 피검체 유래 생물학적 시료에서 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질의 수준을 측정하는 단계를 포함하는, 췌장암의 전이 예측을 위한 정보제공방법을 제공한다.In addition, mRNA of SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) genes in a biological sample derived from a subject or encoded from the gene It provides an information providing method for predicting metastasis of pancreatic cancer, including the step of measuring the level of the protein.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.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.
본 발명의 목적을 달성하기 위하여, 본 발명은 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3)을 포함하는 유전자의 mRNA 또는 상기 유전자가 암호화하는 단백질을 포함하는, 췌장암의 전이 예측용 마커 조성물을 제공한다.In order to achieve the object of the present invention, the present invention provides a gene comprising SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) It provides a marker composition for predicting metastasis of pancreatic cancer, comprising the mRNA or protein encoded by the gene.
본 발명의 일 구현예에서, 상기 마커 조성물은 HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) 및 GSDMA (Gasdermin-A; NM_178171.5)로 이루어진 군에서 선택된 하나 이상의 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질을 더 포함할 수 있다.In one embodiment of the present invention, the marker composition is HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 ( Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3 , NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT ( Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3), and GSDMA (Gasdermin-A; NM_178171.5) mRNA of at least one gene selected from the group consisting of, or a protein encoded by the gene may further include.
또한, 본 발명은 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3)을 포함하는 유전자의 mRNA 또는 상기 유전자가 암호화하는 단백질 수준을 측정하는 제제를 포함하는, 췌장암의 전이 예측용 조성물 및 이를 포함하는 췌장암의 전이 예측용 키트를 제공한다. In addition, the present invention provides mRNA of a gene comprising SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) or the gene is encoded by the gene It provides a composition for predicting metastasis of pancreatic cancer, including an agent for measuring the protein level, and a kit for predicting metastasis of pancreatic cancer comprising the same.
본 발명의 일 구현예에서, 상기 췌장암의 전이 예측용 조성물은 HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) 및 GSDMA (Gasdermin-A; NM_178171.5)로 이루어진 군에서 선택된 하나 이상의 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질 수준을 측정하는 제제를 더 포함할 수 있다.In one embodiment of the present invention, the composition for predicting metastasis of pancreatic cancer is HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) ), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3 , NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3) ), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3), and GSDMA (Gasdermin-A; NM_178171.5) mRNA of one or more genes or the gene It may further comprise an agent for measuring the protein level encoded from.
본 발명의 다른 구현예에서, 상기 전이는 간으로의 전이일 수 있다.In another embodiment of the present invention, the metastasis may be metastasis to the liver.
또한, 본 발명은 피검체 유래 생물학적 시료에서 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질의 수준을 측정하는 단계를 포함하는, 췌장암의 전이 예측을 위한 정보제공방법을 제공한다.In addition, the present invention provides mRNA of SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) genes in a subject-derived biological sample or the above It provides an information providing method for predicting metastasis of pancreatic cancer, including the step of measuring the level of a protein encoded by a gene.
본 발명의 일 구현예에서, 상기 방법은 HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) 및 GSDMA (Gasdermin-A; NM_178171.5)로 이루어진 군에서 선택된 하나 이상의 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질 수준을 측정하는 단계를 더 포함할 수 있다.In one embodiment of the present invention, the method comprises HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha) -actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular mRNA of one or more genes selected from the group consisting of glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3), and GSDMA (Gasdermin-A; NM_178171.5), or a protein level encoded by the gene It may further include the step of measuring.
본 발명의 다른 구현예에서, 상기 생물학적 시료는 혈액 또는 혈장 유래 엑소좀일 수 있다.In another embodiment of the present invention, the biological sample may be exosomes derived from blood or plasma.
본 발명의 또 다른 구현예에서, 상기 mRNA 수준은 차세대 염기서열 분석(Next generation sequencing; NGS), 중합효소연쇄반응(PCR), 역전사 중합효소연쇄반응(RT-PCR), 실시간 중합효소연쇄반응(Real-time PCR), RNase 보호 분석법(RNase protection assay; RPA), 마이크로어레이(microarray), 및 노던 블롯팅(northern blotting)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정될 수 있다.In another embodiment of the present invention, the mRNA level is determined by next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction ( Real-time PCR), RNase protection assay (RPA), microarray, and northern blotting (northern blotting) can be measured through one or more methods selected from the group consisting of.
본 발명의 또 다른 구현예에서, 상기 단백질 수준은 웨스턴 블롯팅(western blotting), 방사선면역분석법(radioimmunoassay; RIA), 방사 면역 확산법(radioimmunodiffusion), 효소면역분석법(ELISA), 면역침강법(immunoprecipitation), 유세포분석법(flow cytometry), 면역형광염색법(immunofluorescence), 오우크테로니(ouchterlony), 보체 고정 분석법(complement fixation assay), 및 단백질 칩(protein chip)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정될 수 있다.In another embodiment of the present invention, the protein level is determined by western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation. , flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and at least one method selected from the group consisting of a protein chip can be measured through
본 발명자들은 정상인 및 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 초기 췌장암 환자에 비해 간 전이를 나타내는 췌장암 환자에서 유의적으로 발현이 증가한 유전자를 발굴하였는바, 상기 유전자는 췌장암의 전이 가능성을 예측하기 위한 바이오마커로 유용하게 이용될 수 있을 것으로 기대된다.The present inventors analyzed the protein in the exosomes isolated from the plasma of normal persons and pancreatic cancer patients, and discovered a gene whose expression was significantly increased in pancreatic cancer patients showing liver metastasis compared to early pancreatic cancer patients. It is expected that it will be usefully used as a biomarker for predicting potential.
도 1 내지 11은 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석한 것으로 도면에 나타난 막대는 각 샘플의 정량적 단백질 양을 나타내며, 그 중 파란색 막대는 각 그룹 샘플의 평균값과 표준편차를 나타낸 것이다(모든 그래프의 x축은 Quan Channels로서 좌측은 No Mota, 우측은 Liver-Mota를 나타내며, y축은 Abundance [a.u.]로서 눈금당 10을 나타낸다).1 to 11 are analyzes of proteins in exosomes isolated from plasma of patients with early pancreatic cancer and pancreatic cancer patients showing liver metastasis. The bars in the figure represent the quantitative protein amounts of each sample, of which blue bars are samples of each group. (The x-axis of all graphs is Quan Channels, the left is No Mota, the right is Liver-Mota, and the y-axis is Abundance [a.u.], which is 10 per division).
도 1은 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2)의 발현 수준이 증가하는 것을 나타낸 결과이다.1 is an analysis of the protein in the exosomes isolated from plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly SERPINB12 (Isoform 2 of Serpin B12; NCBI accession) number in pancreatic cancer patients showing liver metastasis. : NM_001307928.2)) is a result showing an increase in the expression level.
도 2는 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 MMRN1 (Multimerin-1; NCBI 접근(accession)번호 : NM_001371403.1, NM_007351.3)의 발현 수준이 증가하는 것을 나타낸 결과이다.2 is an analysis of the protein in the exosomes isolated from plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly in pancreatic cancer patients showing liver metastasis MMRN1 (Multimerin-1; NCBI accession number: NM_001371403) .1, the result showing that the expression level of NM_007351.3) increases.
도 3은 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 HPSE (Heparanase; NCBI 접근(accession)번호 : NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1)의 발현 수준이 증가하는 것을 나타낸 결과이다.3 is an analysis of proteins in exosomes isolated from plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly HPSE (Heparanase; NCBI accession number: NM_006665.6 in pancreatic cancer patients showing liver metastasis) , NM_001098540.3, NM_001166498.3, NM_001199830.1) show that the expression level is increased.
도 4는 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 AZGP1 (Zinc-alpha-2-glycoprotein; NCBI 접근(accession)번호 : NM_001185.4)의 발현 수준이 증가하는 것을 나타낸 결과이다.4 is an analysis of the protein in the exosomes isolated from plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly AZGP1 (Zinc-alpha-2-glycoprotein; NCBI accession (accession) in pancreatic cancer patients showing liver metastasis. ) No.: This is a result showing that the expression level of NM_001185.4) increases.
도 5는 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 ACTN2 (Alpha-actinin-2; NCBI 접근(accession)번호 : NM_001103.4, NM_001278343.2, NM_001278344.2)의 발현 수준이 증가하는 것을 나타낸 결과이다.5 is an analysis of the proteins in the exosomes isolated from plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly ACTN2 (Alpha-actinin-2; NCBI accession) in pancreatic cancer patients showing liver metastasis. : NM_001103.4, NM_001278343.2, NM_001278344.2) expression level is increased.
도 6은 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 APP (Amyloid-beta precursor protein; NCBI 접근(accession)번호 : NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1)의 발현 수준이 증가하는 것을 나타낸 결과이다.6 is an analysis of the protein in the exosomes isolated from the plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly APP (Amyloid-beta precursor protein; NCBI accession) number in pancreatic cancer patients showing liver metastasis. : NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1) expression level increased It is a result that shows
도 7은 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 ARG1 (Isoform 2 of Arginase-1; NCBI 접근(accession)번호 : NM_001244438.2)의 발현 수준이 증가하는 것을 나타낸 결과이다.7 is an analysis of the protein in the exosomes isolated from plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly ARG1 (Isoform 2 of Arginase-1; NCBI access) in pancreatic cancer patients showing liver metastasis No.: NM_001244438.2) is a result showing an increase in the expression level.
도 8은 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 MMP9 (Matrix metalloproteinase-9; NCBI 접근(accession)번호 : NM_004994.3)의 발현 수준이 증가하는 것을 나타낸 결과이다.8 is an analysis of proteins in exosomes isolated from plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly MMP9 (Matrix metalloproteinase-9; NCBI accession) number in pancreatic cancer patients showing liver metastasis: NM_004994.3) is a result showing an increase in the expression level.
도 9는 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 LACRT (Extracellular glycoprotein lacritin; NCBI 접근(accession)번호 : NM_033277.2)의 발현 수준이 증가하는 것을 나타낸 결과이다.9 is an analysis of the protein in the exosomes isolated from plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly in pancreatic cancer patients showing liver metastasis LACRT (Extracellular glycoprotein lacritin; NCBI accession number: NM_033277) .2) is a result showing an increase in the expression level.
도 10은 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 KRT3 (Keratin, type II cytoskeletal 3; NCBI 접근(accession)번호 : NM_057088.3)의 발현 수준이 증가하는 것을 나타낸 결과이다.Figure 10 shows that by analyzing the protein in the exosomes isolated from the plasma of the pancreatic cancer patient and the pancreatic cancer patient showing liver metastasis, KRT3 (Keratin, type II cytoskeletal 3; NCBI accession) significantly in the pancreatic cancer patient showing liver metastasis No.: NM_057088.3) is a result showing an increase in the expression level.
도 11은 초기 췌장암 환자 및 간 전이를 나타내는 췌장암 환자의 혈장에서 분리한 엑소좀 내 단백질을 분석하여, 간 전이를 나타내는 췌장암 환자에서 유의적으로 GSDMA (Gasdermin-A; NCBI 접근(accession)번호 : NM_178171.5)의 발현 수준이 증가하는 것을 나타낸 결과이다.11 is an analysis of exosome proteins isolated from plasma of patients with early pancreatic cancer and pancreatic cancer showing liver metastasis, and significantly GSDMA (Gasdermin-A; NCBI accession number: NM_178171 in pancreatic cancer patients showing liver metastasis) .5) is a result showing an increase in the expression level.
본 발명자들은 췌장암의 전이를 예측할 수 있는 바이오마커를 개발하고자 초기 췌장암 환자 및 간전이를 나타낸 췌장암 환자에서 혈장을 채취하고 이로부터 엑소좀(exosome)을 분리하여 엑소좀 단백질을 분석한 결과, 간 전이를 나타내는 췌장암 환자에서만 유의하게 발현이 증가하는 유전자를 발견하였는바, 이에 기초하여 본 발명을 완성하였다.In order to develop a biomarker that can predict the metastasis of pancreatic cancer, the present inventors collected plasma from patients with early pancreatic cancer and pancreatic cancer patients who showed liver metastasis, and separated exosomes from these to analyze exosome proteins. As a result, liver metastasis The present invention was completed based on the discovery of a gene whose expression was significantly increased only in pancreatic cancer patients showing
이에, 본 발명은 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3)을 포함하는 유전자의 mRNA 또는 상기 유전자가 암호화하는 단백질을 포함하는, 췌장암의 전이 예측용 마커 조성물을 제공한다.Accordingly, the present invention provides mRNA of a gene comprising SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) or the gene is encoded by the gene It provides a marker composition for predicting metastasis of pancreatic cancer, including a protein.
본 발명에서 사용되는 용어 “췌장암”은 췌장 세포가 정상적인 성장한계를 무시하고 분열 및 성장하는 공격(aggressive) 특성, 주위 조직에 침투하는 침투적(invasive) 특성 및 체내의 다른 부위로 퍼지는 전이적(metastatic) 특성을 갖는 세포에 의한 질병을 총칭하는 의미이다. As used herein, the term “pancreatic cancer” refers to an aggressive characteristic in which pancreatic cells divide and grow ignoring normal growth limits, an invasive characteristic that penetrates into surrounding tissues, and a metastatic (metastatic) characteristic that spreads to other parts of the body. Metastatic) refers to diseases caused by cells with characteristics.
본 발명에서 사용되는 용어, “전이 예측”이란 원발성 암에서 암세포들이 림프를 타고 이동하여 다른 조직으로 이전되는 것을 예측하는 것을 의미한다. 본 발명에서의 전이 예측은 췌장암 세포가 다른 조직으로 이동하는 것을 의미하며, 간 조직으로의 전이인 것이 바람직하나 이에 제한되는 것은 아니다. As used herein, the term “prediction of metastasis” means predicting that cancer cells in primary cancer move through lymph and migrate to other tissues. Prediction of metastasis in the present invention means that pancreatic cancer cells move to another tissue, and metastasis to liver tissue is preferable, but is not limited thereto.
본 발명에 있어서, 상기 마커 조성물은 HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) 및 GSDMA (Gasdermin-A; NM_178171.5)로 이루어진 군에서 선택된 하나 이상의 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질을 더 포함할 수 있다.In the present invention, the marker composition is HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin) -2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131. 3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin) ; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) and GSDMA (Gasdermin-A; NM_178171.5) further comprising mRNA of one or more genes selected from the group consisting of or a protein encoded by the gene can do.
본 발명자들은 구체적인 실시예를 통해 췌장암의 전이 예측을 위한 신규 마커로써 본 발명에 따른 특정 유전자의 용도를 규명하였다.The present inventors identified the use of a specific gene according to the present invention as a novel marker for the prediction of pancreatic cancer metastasis through specific examples.
본 발명의 일 실시예에서는 초기 췌장암 환자에 비해 간 전이를 나타내는 췌장암 환자의 검체에서 특정 유전자(SERPINB12, MMRN1, HPSE, AZGP1, ACTN2, APP, ARG1, MMP9, LACRT, KRT3 및 GSDMA)의 발현이 유의하게 증가되는 것을 확인하였다(실시예 2 참조).In one embodiment of the present invention, the expression of specific genes (SERPINB12, MMRN1, HPSE, AZGP1, ACTN2, APP, ARG1, MMP9, LACRT, KRT3 and GSDMA) in a sample of a pancreatic cancer patient showing liver metastasis compared to an early pancreatic cancer patient is significant It was confirmed that the increase was significantly increased (see Example 2).
상기 결과를 통해 특정 유전자(SERPINB12, MMRN1, HPSE, AZGP1, ACTN2, APP, ARG1, MMP9, LACRT, KRT3 및 GSDMA) 또는 상기 유전자가 암호화하는 단백질이 췌장암의 전이 예측을 위한 마커로 이용될 수 있음을 유추할 수 있다. Through the above results, it was confirmed that specific genes (SERPINB12, MMRN1, HPSE, AZGP1, ACTN2, APP, ARG1, MMP9, LACRT, KRT3 and GSDMA) or proteins encoded by the genes can be used as markers for predicting metastasis of pancreatic cancer can be inferred.
본 발명의 다른 양태로서, 본 발명은 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3)을 포함하는 유전자의 mRNA 또는 상기 유전자가 암호화하는 단백질 수준을 측정하는 제제를 포함하는, 췌장암의 전이 예측용 조성물 및 상기 조성물을 포함하는 췌장암의 전이 예측용 키트를 제공한다.As another aspect of the present invention, the present invention provides mRNA of a gene comprising SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) Or it provides a composition for predicting metastasis of pancreatic cancer, and a kit for predicting metastasis of pancreatic cancer comprising the composition, comprising an agent for measuring the level of the protein encoded by the gene.
본 발명에 있어서, 상기 췌장암의 전이 예측용 조성물은 HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) 및 GSDMA (Gasdermin-A; NM_178171.5)으로 이루어진 군에서 선택된 하나 이상의 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질 수준을 측정하는 제제를 더 포함할 수 있다.In the present invention, the composition for predicting metastasis of pancreatic cancer is HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130. 3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) and GSDMA (Gasdermin-A; NM_178171.5) mRNA of one or more genes selected from the group consisting of or encoded by the gene It may further include an agent for measuring the protein level.
본 발명의 예측용 키트는 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성성분 조성물, 용액 또는 장치로 구성된다.The kit for prediction of the present invention consists of one or more other component compositions, solutions or devices suitable for the analysis method.
예컨대, 본 발명의 키트는 PCR을 수행하기 위해, 분석하고자 하는 시료로부터 유래된 게놈 DNA, 본 발명의 마커 유전자에 대해 특이적인 프라이머 세트, 적당량의 DNA 중합 효소, dNTP 혼합물, PCR 완충용액 및 물을 포함하는 키트일 수 있다. 상기 PCR 완충용액은 KCl, Tris-HCl 및 MgCl2를 함유할 수 있다. 이외에 PCR 산물의 증폭 여부를 확인할 수 있는 전기영동 수행에 필요한 구성 성분들이 본 발명의 키트에 추가로 포함될 수 있다.For example, the kit of the present invention contains genomic DNA derived from a sample to be analyzed, a primer set specific for the marker gene of the present invention, an appropriate amount of a DNA polymerase, a dNTP mixture, a PCR buffer, and water to perform PCR. It may be a kit comprising The PCR buffer may contain KCl, Tris-HCl and MgCl2. In addition, components necessary for performing electrophoresis that can confirm whether or not the PCR product is amplified may be additionally included in the kit of the present invention.
또한, 본 발명의 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. RT-PCR 키트는 마커 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액, 데옥시뉴클레오티드(dNTPs), Taq-폴리머레이즈 및 역전사 효소와 같은 효소, DNase, RNase 억제제, DEPC-수(DEPC-water), 멸균수 등을 포함할 수 있다. 또한 정량 대조군으로 사용되는 유전자에 특이적인 프라이머 쌍을 포함할 수 있다.In addition, the kit of the present invention may be a kit including essential elements necessary for performing RT-PCR. In addition to each primer pair specific for a marker gene, the RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC -Water (DEPC-water), sterile water, etc. may be included. In addition, a primer pair specific for a gene used as a quantitative control may be included.
또한, 본 발명의 키트는 DNA 칩을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. DNA 칩 키트는, 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판을 포함하고, 기판은 정량구조 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있다. 또한, 본 발명의 키트는 본 발명의 마커 유전자가 고정화되어 있는 기판을 갖는 마이크로어레이 형태일 수 있다.In addition, the kit of the present invention may be a kit including essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA corresponding to a gene or fragment thereof is attached as a probe, and the substrate may include cDNA corresponding to a quantitative structural gene or fragment thereof. In addition, the kit of the present invention may be in the form of a microarray having a substrate on which the marker gene of the present invention is immobilized.
또한, 본 발명의 키트는 ELISA를 수행하기 위해 필요한 필수 요소를 포함하는 것을 특징으로 하는 키트일 수 있다. ELISA 키트는 마커 단백질에 대한 특이적인 항체를 포함하며, 상기 단백질 수준을 측정하는 제제를 포함한다. 상기 ELISA 키트는 "항원-항체 복합체"를 형성한 항체를 검출할 수 있는 시약, 예를 들면 표지된 2차 항체, 발색단(chromopores), 효소, 및 그의 기질을 포함할 수 있다. 또한, 정량 대조군 단백질에 특이적인 항체를 포함할 수 있다.In addition, the kit of the present invention may be a kit characterized in that it includes essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific for a marker protein, and an agent for measuring the protein level. The ELISA kit may include a reagent capable of detecting an antibody that has formed an "antigen-antibody complex", for example, a labeled secondary antibody, chromopores, an enzyme, and a substrate thereof. In addition, an antibody specific for the quantitative control protein may be included.
본 발명의 또 다른 양태로서, 본 발명은 피검체 유래 생물학적 시료에서 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질의 수준을 측정하는 단계를 포함하는, 췌장암의 전이 예측을 위한 정보제공방법을 제공한다.As another aspect of the present invention, the present invention provides SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) in a biological sample derived from a subject. ) It provides an information providing method for predicting metastasis of pancreatic cancer, comprising measuring the level of the mRNA of the gene or the protein encoded from the gene.
본 발명에 있어서, 상기 방법은 HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) 및 GSDMA (Gasdermin-A; NM_178171.5)로 이루어진 군에서 선택된 하나 이상의 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질 수준을 측정하는 단계를 더 포함할 수 있다.In the present invention, the method is HPSE (Heparanase; NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin- 2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3 , NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) and GSDMA (Gasdermin-A; NM_178171.5) of one or more genes selected from the group consisting of mRNA or protein levels encoded by the gene It may include further steps.
본 발명에서 사용되는 용어 “췌장암의 전이 예측을 위한 정보제공방법”은 췌장암의 전이 가능성을 예측하기 위한 예비적 단계로써 췌장암의 전이 여부 진단을 위하여 필요한 객관적인 기초정보를 제공하는 것이며 의사의 임상학적 판단 또는 소견은 제외된다. The term “information providing method for predicting metastasis of pancreatic cancer” used in the present invention is a preliminary step for predicting metastasis of pancreatic cancer, and provides objective basic information necessary for diagnosing metastasis of pancreatic cancer, and clinical judgment of doctors or opinions are excluded.
상기 피검체 유래의 생물학적 시료는 조직, 세포, 전혈, 혈액, 타액, 객담, 뇌척수액 및 뇨 등을 포함할 수 있으며, 바람직하게는 혈액 또는 혈장일 수 있고, 더욱 바람직하게는 혈장 유래 엑소좀일 수 있으나 이에 제한되는 것은 아니다.The subject-derived biological sample may include tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid and urine, preferably blood or plasma, and more preferably plasma-derived exosomes. However, the present invention is not limited thereto.
상기 mRNA 수준은 차세대 염기서열 분석(Next generation sequencing; NGS), 중합효소연쇄반응(PCR), 역전사 중합효소연쇄반응(RT-PCR), 실시간 중합효소연쇄반응(Real-time PCR), RNase 보호 분석법(RNase protection assay; RPA), 마이크로어레이(microarray), 및 노던 블롯팅(northern blotting)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정되는 것일 수 있으나, 이에 제한되는 것은 아니다. The mRNA level is determined by next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase protection assay; RPA), microarray (microarray), and may be measured through one or more methods selected from the group consisting of northern blotting (northern blotting), but is not limited thereto.
상기 단백질 수준은 웨스턴 블롯팅(western blotting), 방사선면역분석법(radioimmunoassay; RIA), 방사 면역 확산법(radioimmunodiffusion), 효소면역분석법(ELISA), 면역침강법(immunoprecipitation), 유세포분석법(flow cytometry), 면역형광염색법(immunofluorescence), 오우크테로니(ouchterlony), 보체 고정 분석법(complement fixation assay), 및 단백질 칩(protein chip)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정되는 것일 수 있으나, 이에 제한되는 것은 아니다. The protein level was determined by western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry, and immunity. It may be measured by at least one method selected from the group consisting of immunofluorescence, ouchterlony, complement fixation assay, and protein chip, but is limited thereto it is not going to be
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.
[실시예][Example]
실시예 1. 혈장 내 엑소좀의 특이 단백질 분석Example 1. Analysis of specific proteins of exosomes in plasma
1-1. 엑소좀 샘플 준비1-1. Exosome Sample Preparation
정상인 및 췌장암 환자의 혈액을 BD Vacutainer Plastic K2EDTA Tube (BD, Cat.no 367525)에 채취한 후 2시간 이내 2,500 rpm, 4 ℃ 조건에서 20분간 원심 분리하여 혈장을 분리한다. 상층 혈장을 Protein LoBind Tube (Eppendorf, Cat.no 0030108116)에 500 ㎕씩 분주하고 -80 ℃에 보관하였다. Blood from normal and pancreatic cancer patients is collected in BD Vacutainer Plastic K2EDTA Tube (BD, Cat.no 367525), and plasma is separated by centrifugation within 2 hours at 2,500 rpm and 4°C for 20 minutes. The supernatant plasma was aliquoted into Protein LoBind Tubes (Eppendorf, Cat.no 0030108116) by 500 μl and stored at -80°C.
혈장 시료를 3,000 g, 4 ℃ 조건에서 15분간 원심 분리하여 혈장 내 세포 및 세포 찌꺼기(cell debris)를 제거하였다. 다음 혈장 시료 500 ㎕에 Exo2DTM for Protein assay (EXOSOMEplus) 250 ㎕, 2:1 비율로 첨가하여 Aqueous Two-Phase System을 기반으로 엑소좀을 분리 후, Halt™ Protease and Phosphatase Inhibitor Cocktail, EDTA-Free(Thermo scientific)을 첨가한 200 ㎕ 용량의 PBS에 엑소좀을 재부유(Resuspending)시켜 엑소좀 샘플을 준비하였다. 시료 분석 전까지 -80 ℃에 보관하였다. Plasma samples were centrifuged at 3,000 g and 4 °C for 15 minutes to remove cells and cell debris in the plasma. Then, 250 μl of Exo2DTM for Protein assay (EXOSOMEplus) was added to 500 μl of plasma sample at a ratio of 2: 1 to isolate exosomes based on the Aqueous Two-Phase System, Halt™ Protease and Phosphatase Inhibitor Cocktail, EDTA-Free (Thermo scientific) was added to 200 μl of exosomes in PBS to prepare exosome samples by resuspending them. Samples were stored at -80 °C until analysis.
1-2. 엑소좀 단백질 정량분석1-2. Exosome protein quantitative analysis
상기 실시예 1-1에서 제조된 혈장 엑소좀 샘플 30 ㎕에 프로테아제 및 포스페타아제(1X)를 포함하는 RIPA 버퍼 30 ㎕를 첨가하고 충분히 혼합시킨 후 원심분리 하였다. 추출된 엑소좀 단백질은 Bovine Serum Albumin (BSA) Standard Set (BIO-RAD)와 PierceTM BCA Protein Assay (Thermo scientific)을 이용하여 농도 측정 및 정량분석을 진행하였다.30 μl of RIPA buffer containing protease and phosphatase (1X) was added to 30 μl of the plasma exosome sample prepared in Example 1-1, mixed sufficiently, and centrifuged. The extracted exosome protein was subjected to concentration measurement and quantitative analysis using Bovine Serum Albumin (BSA) Standard Set (BIO-RAD) and PierceTM BCA Protein Assay (Thermo scientific).
다음 엑소좀 단백질 20 ㎍을 SDS loading buffer와 섞은 후 10% Sodium Dodecyl Sulfate Polyacrylamide Gel Eletrophoresis (SDS-PAGE) 기법으로 전기영동 하여 단백질들을 분자량 크기에 따라 분리시켰다. 전기영동된 젤을 InstantBlue™ (Coomassie blue stanning) 시약에 담가 상온에서 15분간 염색한 후 1차적으로 단백질 검출 및 분포 상황을 확인하였다.Then, 20 μg of exosomal protein was mixed with SDS loading buffer and electrophoresed using 10% Sodium Dodecyl Sulfate Polyacrylamide Gel Eletrophoresis (SDS-PAGE) technique to separate proteins according to molecular weight size. After immersing the electrophoresed gel in InstantBlue™ (Coomassie blue stanning) reagent and staining at room temperature for 15 minutes, protein detection and distribution were first checked.
1-3. S-Trap™ Mini Spin Column (ProtiFi)을 이용한 단백질 digestion1-3. Protein digestion using S-Trap™ Mini Spin Column (ProtiFi)
(1) 시료에 5% SDS, 50 mM TAEB (pH7.55), 1x PPIC을 추가하여 용해하였다. 인산을 이용하여 pH 값을 맞춰주었다.(1) 5% SDS, 50 mM TAEB (pH7.55), and 1x PPIC were added to the sample and dissolved. The pH value was adjusted using phosphoric acid.
(2) BCA assay을 이용하여 정량한 단백질 시료 300 ㎍을 취하였다. Digestion할 단백질에 최종농도가 20 mM이 되게 DTT를 추가하여 95 ℃에서 10분 동안 알킬화시켰다.(2) 300 ㎍ of a protein sample quantified using BCA assay was taken. DTT was added to the protein to be digested to a final concentration of 20 mM and alkylated at 95 °C for 10 minutes.
(3) 실온에서 식혀준 후 최종농도가 40 mM 되게 IAA을 추가하여 어두운 곳에서 30분간 reduction 시켰다. 다음 12% 인산을 1.2% 인산으로 되게끔 섞어주었다. (3) After cooling at room temperature, IAA was added so that the final concentration was 40 mM, and reduced for 30 minutes in a dark place. Then, 12% phosphoric acid was mixed to make 1.2% phosphoric acid.
(4) S-Trap binding buffer (90% methanol 100 mM TEAB (pH 7.1)) 350 ㎕ 추가한 후 모두 S-Trap spin column으로 옮겼다. 다음 원심 분리기를 이용하여 4,000 g에서 30초간 원심 분리하였다.(4) 350 μl of S-Trap binding buffer (90% methanol, 100 mM TEAB (pH 7.1)) was added, and all were transferred to an S-Trap spin column. Then, centrifugation was performed at 4,000 g for 30 seconds using a centrifuge.
(5) S-Trap buffer 400 ㎕을 S-Trap spin column에 추가한 후 원심 분리기를 이용하여 4,000 g에서 30초간 원심 분리하였다. 이를 3회 반복하였다.(5) 400 μl of S-Trap buffer was added to the S-Trap spin column and centrifuged at 4,000 g for 30 seconds using a centrifuge. This was repeated 3 times.
(6) S-Trap spin column을 새로운 2 ml sample tube로 옮긴 후, 단백질과1:25 비율의 trypsin을 50 mM TEAB buffer 125 ㎕에 녹여 S-Trap spin column에 추가하여 37 ℃에서 16시간 동안 incubation 하였다.(6) After transferring the S-Trap spin column to a new 2 ml sample tube, dissolve the protein and trypsin in a ratio of 1:25 in 125 μl of 50 mM TEAB buffer, add it to the S-Trap spin column, and incubate at 37°C for 16 hours. did
(7) 50 mM TEAB buffer를 80 ㎕ 추가하여 1,000 g에서 60초간 원심 분리하였다. 다음 0.2% FA를 80 ㎕ 추가하여 1,000 g에서 60초간 원심 분리하였다. 마지막으로 elution buffer (50% ACN, 0.2% FA)를 80 ㎕ 추가하여 4,000 g에서 60초간 원심 분리하고 용리한 시료를 동결 건조하였다.(7) 80 μl of 50 mM TEAB buffer was added and centrifuged at 1,000 g for 60 seconds. Then, 80 μl of 0.2% FA was added and centrifuged at 1,000 g for 60 seconds. Finally, 80 μl of elution buffer (50% ACN, 0.2% FA) was added, centrifuged at 4,000 g for 60 seconds, and the eluted sample was freeze-dried.
1-4. 프로테오믹스 분석1-4. Proteomics analysis
Digestion 과정을 거쳐 준비된 단백질 펩티드 시료를 Q Exactive Plus LC-MS/MS System 질량분석기(Thermo Scientific)를 이용하여 단백질체 분석을 하였다. 각 시료마다 두 번 반복(duplicate)하여 측정하였고, 기기에서 생성된 데이터는 Proteome DiscovererTM 2.2 (Thermo Scientific) 소프트웨어를 이용하여 데이터베이스 검색, 종합적인 단백질 및 펩티드의 식별, 특성 분석과 정량 분석 및 통계분석을 진행하였다.Protein peptide samples prepared through the digestion process were analyzed for proteomic bodies using Q Exactive Plus LC-MS/MS System mass spectrometer (Thermo Scientific). Each sample was measured in duplicate, and the data generated by the instrument was analyzed using Proteome DiscovererTM 2.2 (Thermo Scientific) software for database search, comprehensive protein and peptide identification, characterization and quantitative analysis and statistical analysis. proceeded.
실시예 2. 간 전이를 나타내는 췌장암 환자에서의 특이 유전자 발현 확인Example 2. Identification of specific gene expression in pancreatic cancer patients with liver metastases
췌장암 기수가 1, 2, 3기에 해당하는 초기 췌장암 환자 5명 및 췌장암 기수가 4기에 해당하는 간 전이 췌장암 환자 4명의 혈장을 수득하고, 실시예 1의 방법을 통해 혈장 엑소좀 단백질을 추출한 후, 췌장암 환자에서 유의적인 발현을 나타내는 유전자를 확인하였다.After obtaining plasma from 5 patients with early pancreatic cancer corresponding to stage 1, 2, and 3 of pancreatic cancer and 4 patients with liver metastasis pancreatic cancer corresponding to stage 4 of pancreatic cancer, extracting plasma exosome proteins through the method of Example 1, Genes showing significant expression in pancreatic cancer patients were identified.
그 결과, 도 1 내지 11에 나타낸 바와 같이 SERPINB12, MMRN1, HPSE, AZGP1, ACTN2, APP, ARG1, MMP9, LACRT, KRT3 및 GSDMA 유전자의 발현이 초기췌장암 환자에 비해 간 전이를 나타내는 췌장암 환자의 검체에서만 유의적으로 증가되어 있는 것을 확인하였다.As a result, as shown in FIGS. 1 to 11, the expression of SERPINB12, MMRN1, HPSE, AZGP1, ACTN2, APP, ARG1, MMP9, LACRT, KRT3, and GSDMA genes was found only in the samples of patients with pancreatic cancer showing liver metastases compared to patients with early pancreatic cancer. It was confirmed that there was a significant increase.
상기 결과로부터, 간 전이를 나타내는 췌장암 환자 유래 혈장 엑소좀에서 검출된 특정 유전자(SERPINB12, MMRN1, HPSE, AZGP1, ACTN2, APP, ARG1, MMP9, LACRT, KRT3 및 GSDMA)가 췌장암 환자의 전이를 예측하는데 유용하게 이용될 수 있을 것으로 판단된다.From the above results, specific genes (SERPINB12, MMRN1, HPSE, AZGP1, ACTN2, APP, ARG1, MMP9, LACRT, KRT3 and GSDMA) detected in plasma exosomes derived from pancreatic cancer patients representing liver metastases are predictive of metastasis in pancreatic cancer patients. It is considered to be useful.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (11)

  1. SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3)을 포함하는 유전자의 mRNA 또는 상기 유전자가 암호화하는 단백질을 포함하는, 췌장암의 전이 예측용 마커 조성물.mRNA of a gene including SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) or a protein encoded by the gene , A marker composition for predicting metastasis of pancreatic cancer.
  2. 제1항에 있어서, The method of claim 1,
    상기 마커 조성물은 HPSE (Heparanase; NCBI 접근(accession)번호 : NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) 및 GSDMA (Gasdermin-A; NM_178171.5)로 이루어진 군에서 선택된 하나 이상의 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질을 더 포함하는 것을 특징으로 하는, 마커 조성물.The marker composition is HPSE (Heparanase; NCBI accession number: NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha) -actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular mRNA of one or more genes selected from the group consisting of glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3), and GSDMA (Gasdermin-A; NM_178171.5) or a protein encoded by the gene Further comprising, a marker composition.
  3. SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3)을 포함하는 유전자의 mRNA 또는 상기 유전자가 암호화하는 단백질 수준을 측정하는 제제를 포함하는, 췌장암의 전이 예측용 조성물.SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) Measure the mRNA level of a gene or protein encoded by the gene A composition for predicting metastasis of pancreatic cancer, comprising a formulation to.
  4. 제3항에 있어서,4. The method of claim 3,
    상기 조성물은 HPSE (Heparanase; NCBI 접근(accession)번호 : NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) 및 GSDMA (Gasdermin-A; NM_178171.5)로 이루어진 군에서 선택된 하나 이상의 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질 수준을 측정하는 제제를 더 포함하는 것을 특징으로 하는, 췌장암의 전이 예측용 조성물.The composition comprises HPSE (Heparanase; NCBI accession number: NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha- actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131 .3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein) lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3), and GSDMA (Gasdermin-A; NM_178171.5) of one or more genes selected from the group consisting of mRNA or protein levels encoded by the gene; A composition for predicting metastasis of pancreatic cancer, characterized in that it further comprises an agent to be measured.
  5. 제3항에 있어서,4. The method of claim 3,
    상기 전이는 간으로의 전이인 것을 특징으로 하는, 조성물.The composition, characterized in that the metastasis to the liver.
  6. 제3항의 조성물을 포함하는, 췌장암의 전이 예측용 키트.A kit for predicting metastasis of pancreatic cancer, comprising the composition of claim 3 .
  7. 피검체 유래 생물학적 시료에서 SERPINB12 (Isoform 2 of Serpin B12; NCBI 접근(accession)번호 : NM_001307928.2) 및 MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질의 수준을 측정하는 단계를 포함하는, 췌장암의 전이 예측을 위한 정보제공방법.mRNA of SERPINB12 (Isoform 2 of Serpin B12; NCBI accession number: NM_001307928.2) and MMRN1 (Multimerin-1; NM_001371403.1, NM_007351.3) genes in a biological sample derived from a subject or a protein encoded by the gene An information providing method for predicting metastasis of pancreatic cancer, comprising the step of measuring the level of.
  8. 제7항에 있어서, 상기 방법은 HPSE (Heparanase; NCBI 접근(accession)번호 : NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3, NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3) 및 GSDMA (Gasdermin-A; NM_178171.5)로 이루어진 군에서 선택된 하나 이상의 유전자의 mRNA 또는 상기 유전자로부터 코딩되는 단백질 수준을 측정하는 단계를 더 포함하는 것을 특징으로 하는, 정보제공방법.According to claim 7, wherein the method is HPSE (Heparanase; NCBI accession number: NM_006665.6, NM_001098540.3, NM_001166498.3, NM_001199830.1), AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) ), ACTN2 (Alpha-actinin-2; NM_001103.4, NM_001278343.2, NM_001278344.2), APP (Amyloid-beta precursor protein; NM_000484.4, NM_001385253.1, NM_201414.3, NM_001136016.3, NM_001136129.3 , NM_001136130.3, NM_001136131.3, NM_001204301.2, NM_001204302.2, NM_001204303.2, NM_001385253.1), ARG1 (Isoform 2 of Arginase-1; NM_001244438.2), MMP9 (Matrix metalloproteinase-9; NM_004994.3) ), LACRT (Extracellular glycoprotein lacritin; NM_033277.2), KRT3 (Keratin, type II cytoskeletal 3; NM_057088.3), and GSDMA (Gasdermin-A; NM_178171.5) mRNA of one or more genes or the gene Information providing method, characterized in that it further comprises the step of measuring the level of the encoded protein from.
  9. 제7항에 있어서,8. The method of claim 7,
    상기 생물학적 시료는 혈액 또는 혈장 유래 엑소좀인 것을 특징으로 하는, 정보제공방법.The biological sample is characterized in that the exosomes derived from blood or plasma, information providing method.
  10. 제7항에 있어서, 8. The method of claim 7,
    상기 mRNA 수준은 차세대 염기서열 분석(Next generation sequencing; NGS), 중합효소연쇄반응(PCR), 역전사 중합효소연쇄반응(RT-PCR), 실시간 중합효소연쇄반응(Real-time PCR), RNase 보호 분석법(RNase protection assay; RPA), 마이크로어레이(microarray), 및 노던 블롯팅(northern blotting)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정되는 것을 특징으로 하는, 정보제공방법.The mRNA level is determined by next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase protection assay; RPA), microarray (microarray), characterized in that measured through one or more methods selected from the group consisting of northern blotting (northern blotting), information providing method.
  11. 제7항에 있어서,8. The method of claim 7,
    상기 단백질 수준은 웨스턴 블롯팅(western blotting), 방사선면역분석법(radioimmunoassay; RIA), 방사 면역 확산법(radioimmunodiffusion), 효소면역분석법(ELISA), 면역침강법(immunoprecipitation), 유세포분석법(flow cytometry), 면역형광염색법(immunofluorescence), 오우크테로니(ouchterlony), 보체 고정 분석법(complement fixation assay), 및 단백질 칩(protein chip)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정되는 것을 특징으로 하는, 정보제공방법.The protein level was determined by western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry, immune Information, characterized in that it is measured by at least one method selected from the group consisting of immunofluorescence, ouchterlony, complement fixation assay, and protein chip. How to provide.
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