KR20220074802A - Combined Biomarkers for Diagnosis of Chronic Hepatic Disease Based on Integrated Transcriptome Analysis and Use Thereof - Google Patents
Combined Biomarkers for Diagnosis of Chronic Hepatic Disease Based on Integrated Transcriptome Analysis and Use Thereof Download PDFInfo
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Abstract
본 발명은 통합전사체분석 기반 만성 간질환 진단용 복합마커 및 이의 용도에 관한 것으로, 보다 상세하게는 지방증, 비알코올성 지방간염 환자를 대상으로 통합전사체 분석 및 머신러닝 분석을 통해 선별한 28종 복합 마커 및 이를 이용한 만성 간질환 진단에 대한 정보를 제공하는 방법에 관한 것이다.
본 발명에서는 지방증, NASH 환자를 대상으로 통합전사제 분석을 수행하여 차별 발현 유전자(Differentially Expressed Gene:DEG) 분석을 통해 선별된 유전자 및 특징 선택(Feature selection) 프로세스를 이용하여 선별된 유전자 중에서 공동으로 선별된 28종의 유전자를 최종 선별하였으며, 28종의 복합마커 또는 ATP8B1(ATPase phospholipid transporting 8B1) 마커를 이용하는 경우 만성 간질환 진단능이 현저하게 향상된 것을 확인하였으므로, 본 발명의 복합마커는 만성 간질환 진단을 위한 바이오마커로 적용할 수 있는 것을 확인하였다.The present invention relates to a complex marker for diagnosing chronic liver disease based on integrated transcriptome analysis and its use, and more particularly, 28 types of complexes selected through integrated transcriptome analysis and machine learning analysis for patients with steatosis and nonalcoholic steatohepatitis It relates to a marker and a method for providing information on the diagnosis of chronic liver disease using the same.
In the present invention, integrated transcriptase analysis is performed on steatosis and NASH patients to jointly select genes selected through differentially expressed gene (DEG) analysis and genes selected using a feature selection process. 28 kinds of selected genes were finally selected, and it was confirmed that the diagnostic ability of chronic liver disease was significantly improved when 28 kinds of complex markers or ATP8B1 (ATPase phospholipid transporting 8B1) markers were used. It was confirmed that it can be applied as a biomarker for
Description
본 발명은 통합전사체분석 기반 만성 간질환 진단용 복합마커 및 이의 용도에 관한 것으로, 보다 상세하게는 지방증, 비알코올성 지방간염 환자를 대상으로 통합전사체 분석 및 머신러닝 분석을 통해 선별한 28종 복합 마커 및 이를 이용한 만성 간질환 진단에 대한 정보를 제공하는 방법에 관한 것이다.The present invention relates to a complex marker for diagnosing chronic liver disease based on integrated transcriptome analysis and its use, and more particularly, 28 types of complexes selected through integrated transcriptome analysis and machine learning analysis for patients with steatosis and nonalcoholic steatohepatitis It relates to a marker and a method for providing information on the diagnosis of chronic liver disease using the same.
만성 간질환으로 인한 국내의 사회경제적 부담은 2010년 기준 약 3조 7000억 원으로 가장 심각한 질환 중 하나이다. 한국에서 발병빈도가 매우 높고 특히 40 ~ 50대의 경우 간암 및 간질환에 의한 사망률이 가장 높은 것으로 알려져 있다.The socioeconomic burden of chronic liver disease in Korea amounted to about 3.7 trillion won as of 2010, making it one of the most serious diseases. It is known that the incidence rate is very high in Korea, and the death rate from liver cancer and liver disease is the highest, especially in those in their 40s and 50s.
만성 간질환 중 하나인 비-알코올성 지방간질환(non-alcoholic fatty liver disease; NAFLD)은 단순한 지방증으로부터 비-알코올성 지방간염((non-alcoholic steatohepatitis; NASH)에 이르는 범위의 진행성 간질환이다. 특히, 비-알코올성 지방간염(NASH)은 조직학적으로 알코올성 간염과 비슷한 지방산 축적, 간세포 손상 및 염증에 의해 특징화되는 간의 진행성 질환으로, 간 지방증으로부터 간경변증 및 간부전으로 확장되는 공정의 주요 단계이며, 최근 NASH의 발병이 최근 여러 해 동안 증가하고 있으며, NASH로 진행되는 환자는 간-관련된 유병율 및 사망률이 증가하고 있는 추세이다.Non-alcoholic fatty liver disease (NAFLD), one of the chronic liver diseases, is a progressive liver disease ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). In particular, Non-alcoholic steatohepatitis (NASH) is a progressive disease of the liver characterized by fatty acid accumulation, hepatocellular damage and inflammation histologically similar to alcoholic hepatitis, a major step in the process extending from hepatic steatosis to cirrhosis and liver failure, and recently NASH The incidence has been increasing in recent years, and the liver-related morbidity and mortality in patients who progress to NASH are increasing.
이런 NASH를 포함하는 만성간질환의 활성, 단계 또는 중증도를 진단하기 위한 표준 방법으로 간생검(liver biopsy) 표본의 조직학적 검사를 사용하고 있으나, 간생검은 침습적 방법이라는 단점을 갖고 있다. 또한, 계속해서 증가하는 간 질환 환자들을 대상으로 모두 조직검사를 수행하는 것은 여러 가지 한계가 있고, 간생검은 통증, 출혈 및 아주 드문 경우 사망에 이를 수도 있는 부작용이 있다 (Rana L Smalling et al., Am J Physiol Gastrointest Liver Physiol., 305(5):G364-74, 2013; 대한민국공개특허 제 10-2020-0051676호).Although the histological examination of a liver biopsy specimen is used as a standard method for diagnosing the activity, stage, or severity of chronic liver disease including NASH, the liver biopsy has the disadvantage of being an invasive method. In addition, performing a biopsy for all patients with liver disease, which is constantly increasing, has several limitations, and liver biopsy has side effects that can lead to pain, bleeding, and, in very rare cases, death (Rana L Smalling et al. , Am J Physiol Gastrointest Liver Physiol. , 305(5):G364-74, 2013; Korean Patent Publication No. 10-2020-0051676).
따라서, 침습적인 간생검을 대체할 수 있는 신뢰할만한 진단방법의 개발이 요구되고 있으며, 이를 위해 다양한 간질환 진단용 바이오마커가 발굴 및 연구되고 있다Therefore, the development of a reliable diagnostic method that can replace the invasive liver biopsy is required, and for this purpose, various biomarkers for diagnosing liver disease are being discovered and studied.
이에, 본 발명에서는 보다 효과적인 만성 간질환 진단용 바이오마커를 개발하기 위해 노력한 결과, 지방증과 NASH 환자 그룹을 대상으로 통합전사체 분석을 수행하여 선별된 유전자 및 다양한 분석방법을 통한 특징 선택(Feature selection) 프로세스를 이용하여 선별된 유전자 중에서 공동으로 선별된 28종의 유전자를 최종 선별하였으며, 28종의 복합마커 또는 ATP8B1 마커를 이용하는 경우 만성 간질환 진단능이 현저하게 향상된 것을 확인하고, 본 발명을 완성하였다.Accordingly, in the present invention, as a result of efforts to develop a more effective biomarker for diagnosing chronic liver disease, integrated transcriptome analysis was performed on a group of steatosis and NASH patients to select genes and feature selection through various analysis methods. 28 types of genes jointly selected from among the genes selected using the process were finally selected, and when 28 types of complex markers or ATP8B1 markers were used, it was confirmed that the diagnostic ability of chronic liver disease was remarkably improved, and the present invention was completed.
본 발명의 목적은 28종의 마커를 포함하는 만성 간질환 진단용 복합마커 조성물 및 복합마커의 mRNA 또는 단백질 수준을 측정하는 제제를 포함하는 만성간질환 진단용 조성물을 제공하는 데 있다.It is an object of the present invention to provide a composition for diagnosing chronic liver disease, comprising a complex marker composition for diagnosing chronic liver disease including 28 markers and an agent for measuring mRNA or protein levels of the complex marker.
본 발명의 다른 목적은 상기 만성 간질환 진단용 조성물을 포함하는 만성 간질환 진단용 키트를 제공하는 데 있다.Another object of the present invention is to provide a kit for diagnosing chronic liver disease comprising the composition for diagnosing chronic liver disease.
본 발명의 또 다른 목적은 상기 만성 간질환 진단용 조성물을 이용한 만성 간질환 진단을 위한 정보 제공방법을 제공하는 데 있다.Another object of the present invention is to provide a method for providing information for diagnosing chronic liver disease using the composition for diagnosing chronic liver disease.
상기 목적을 달성하기 위해, In order to achieve the above purpose,
본 발명은 AJUBA(ajuba LIM protein), ANXA2(annexin A2), ATP8B1(ATPase phospholipid transporting 8B1), CCND1(cyclin D1), CEBPD(CCAAT enhancer binding protein), CHST9(carbohydrate sulfotransferase 9), CLDN7(claudin 7), DNAJC12(DnaJ heat shock protein family (Hsp40) member C12), DPYSL2(dihydropyrimidinase like 2), FAT1(FAT atypical cadherin 1), GPNMB(glycoprotein nmb), ITGA6(integrin subunit alpha 6), KRT8(keratin 8), LGALS3(galectin 3), MCM3(minichromosome maintenance complex component 3), MCM4(minichromosome maintenance complex component 4), MCM6(minichromosome maintenance complex component 6), MSN(moesin), NR0B2(nuclear receptor subfamily 0 group B member 2), PACSIN3(protein kinase C and casein kinase substrate in neurons 3), PDGFRA(platelet derived growth factor receptor alpha), PIK3IP1(phosphoinositide-3-kinase interacting protein 1), RNASE1(ribonuclease A family member 1, pancreatic), RNF152(ring finger protein 152), SFXN2(sideroflexin 2), SULF2(sulfatase 2), TTC36(tetratricopeptide repeat domain 36) 및 WIPI1(WD repeat domain, phosphoinositide interacting 1)을 포함하는 복합마커; 또는 The present invention is AJUBA (ajuba LIM protein), ANXA2 (annexin A2), ATP8B1 (ATPase phospholipid transporting 8B1), CCND1 (cyclin D1), CEBPD (CCAAT enhancer binding protein), CHST9 (carbohydrate sulfotransferase 9), CLDN7 (claudin 7) , DNAJC12 (DnaJ heat shock protein family (Hsp40) member C12), DPYSL2 (dihydropyrimidinase like 2), FAT1 (FAT atypical cadherin 1), GPNMB (glycoprotein nmb), ITGA6 (integrin subunit alpha 6), KRT8 (keratin 8), LGALS3 (galectin 3), MCM3 (minichromosome maintenance complex component 3), MCM4 (minichromosome maintenance complex component 4), MCM6 (minichromosome maintenance complex component 6), MSN (moesin), NR0B2 (
ATP8B1(ATPase phospholipid transporting 8B1) 마커를 포함하는 만성 간질환 진단 마커 조성물을 제공한다.Provided is a chronic liver disease diagnostic marker composition comprising an ATPase phospholipid transporting 8B1 (ATP8B1) marker.
또한, 본 발명은 상기 만성 간질환 진단 마커 조성물의 mRNA 또는 단백질 수준을 측정하는 제제를 포함하는 만성 간질환 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing chronic liver disease comprising an agent for measuring the mRNA or protein level of the chronic liver disease diagnostic marker composition.
본 발명의 바람직한 일실시예에 있어서, 상기 만성 간질환은 지방증, 비-알코올성 지방간질환(non-alcoholic fatty liver disease; NAFLD), 비-알코올성 지방간염((non-alcoholic steatohepatitis; NASH) 또는 간경화증일 수 있다.In a preferred embodiment of the present invention, the chronic liver disease is steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) or liver cirrhosis. can
본 발명의 바람직한 다른 일실시예에 있어서, 상기 mRNA 수준을 측정하는 제제는 마커에 특이적으로 결합하는 프라이머쌍, 프로브 또는 안티센스 뉴클레오타이드일 수 있다.In another preferred embodiment of the present invention, the agent for measuring the mRNA level may be a primer pair, a probe, or an antisense nucleotide that specifically binds to a marker.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 단백질 수준을 측정하는 제제는 마커 또는 마커의 펩타이드 단편에 특이적으로 결합하는 항체, 상호작용 단백질, 리간드, 나노입자(nanoparticles) 또는 압타머(aptamer)를 포함할 수 있다. In another preferred embodiment of the present invention, the agent for measuring the protein level is an antibody that specifically binds to a marker or a peptide fragment of a marker, an interacting protein, a ligand, nanoparticles, or an aptamer ) may be included.
또한, 본 발명은 상기 만성 간질환 진단용 조성물을 포함하는 만성 간질환 진단용 키트를 제공한다.In addition, the present invention provides a kit for diagnosing chronic liver disease comprising the composition for diagnosing chronic liver disease.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 키트는 RT-PCR(Reverse transcription polymerase chain reaction) 키트, DNA 칩 키트, ELISA(enzyme linked immunosorbent assay) 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(Multiple reaction monitoring) 키트일 수 있다.In another preferred embodiment of the present invention, the kit is a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an ELISA (enzyme linked immunosorbent assay) kit, a protein chip kit, a rapid kit or It may be a multiple reaction monitoring (MRM) kit.
또한, 본 발명은 (a) 환자의 생물학적 시료로부터 ATP8B1 마커 또는 상기 복합마커의 mRNA 또는 단백질 발현 수준을 측정하는 단계; 및 In addition, the present invention comprises the steps of (a) measuring the mRNA or protein expression level of the ATP8B1 marker or the complex marker from the patient's biological sample; and
(b) 상기 mRNA 또는 단백질 발현 수준을 대조군 시료와 비교하는 단계를 포함하는 만성 간질환 진단을 위한 정보 제공방법을 제공한다.(b) provides a method for providing information for diagnosing chronic liver disease, comprising comparing the mRNA or protein expression level with a control sample.
본 발명의 바람직한 일실시예에 있어서, 상기 만성 간질환 진단을 위한 정보 제공방법은 CEBPD(CCAAT enhancer binding protein), DNAJC12(DnaJ heat shock protein family (Hsp40) member C12), NR0B2(nuclear receptor subfamily 0 group B member 2), PACSIN3(protein kinase C and casein kinase substrate in neurons 3), RNF152(ring finger protein 152) 및 SFXN2(sideroflexin 2), TTC36(tetratricopeptide repeat domain 36) 마커의 mRNA 또는 이의 단백질 발현 수준이 대조군에 비해 감소하고,In a preferred embodiment of the present invention, the information providing method for diagnosing chronic liver disease is CEBPD (CCAAT enhancer binding protein), DNAJC12 (DnaJ heat shock protein family (Hsp40) member C12), NR0B2 (
AJUBA(ajuba LIM protein), ANXA2(annexin A2), ATP8B1(ATPase phospholipid transporting 8B1), CCND1(cyclin D1), CHST9(carbohydrate sulfotransferase 9), CLDN7(claudin 7), DPYSL2(dihydropyrimidinase like 2), FAT1(FAT atypical cadherin 1), GPNMB(glycoprotein nmb), ITGA6(integrin subunit alpha 6), KRT8(keratin 8), LGALS3(galectin 3), MCM3(minichromosome maintenance complex component 3), MCM4(minichromosome maintenance complex component 4), MCM6(minichromosome maintenance complex component 6), MSN(moesin), PDGFRA(platelet derived growth factor receptor alpha), PIK3IP1(phosphoinositide-3-kinase interacting protein 1), RNASE1(ribonuclease A family member 1, pancreatic), SULF2(sulfatase 2), 및 WIPI1(WD repeat domain, phosphoinositide interacting 1) 마커의 mRNA 또는 이의 단백질 발현 수준이 대조군에 비해 증가하면, 만성 간질환인 것으로 정보를 제공하는 단계를 추가로 포함할 수 있다.AJUBA (ajuba LIM protein), ANXA2 (annexin A2), ATP8B1 (ATPase phospholipid transporting 8B1), CCND1 (cyclin D1), CHST9 (carbohydrate sulfotransferase 9), CLDN7 (claudin 7), DPYSL2 (dihydropyrimidinase like 2), FAT1 (FAT1) atypical cadherin 1), GPNMB (glycoprotein nmb), ITGA6 (integrin subunit alpha 6), KRT8 (keratin 8), LGALS3 (galectin 3), MCM3 (minichromosome maintenance complex component 3), MCM4 (minichromosome maintenance complex component 4), MCM6 (minichromosome maintenance complex component 6), MSN (moesin), PDGFRA (platelet derived growth factor receptor alpha), PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), RNASE1 (ribonuclease
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 만성 간질환 진단을 위한 정보 제공방법은 ATP8B1(ATPase phospholipid transporting 8B1) 마커의 mRNA 또는 이의 단백질 발현 수준이 대조군에 비해 증가하면, 만성 간질환인 것으로 정보를 제공하는 단계를 추가로 포함할 수 있다.In another preferred embodiment of the present invention, the information providing method for diagnosing chronic liver disease is when the mRNA or protein expression level of ATP8B1 (ATPase phospholipid transporting 8B1) marker increases compared to the control, chronic liver disease. The step of providing information may be further included.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 mRNA 발현 정도 측정은 역전사효소 중합효소반응, 경쟁적 역전사효소 중합효소반응, 실시간 역전사효소 중합효소반응, RNase 보호 분석법, 노던 블랏팅 또는 DNA 칩을 이용하여 수행할 수 있다. In another preferred embodiment of the present invention, the mRNA expression level is measured using a reverse transcriptase polymerase reaction, a competitive reverse transcriptase polymerase reaction, a real-time reverse transcriptase polymerase reaction, an RNase protection assay, Northern blotting or a DNA chip. can be done by
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 단백질 발현 정도 측정은 단백질 칩 분석, 면역측정법, 리간드 바인딩 어세이, MALDI-TOF(Matrix Desorption/Ionization Time of Flight Mass Spectrometry)분석, SELDI-TOF(Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry)분석, 방사선 면역분석, 방사 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 보체 고정 분석법, 2차원 전기영동 분석, 액상 크로마토그래피-질량분석(liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS(liquid chromatography-Mass Spectrometry/ Mass Spectrometry), 웨스턴 블랏 및 ELISA(enzyme linked immunosorbent assay)를 이용하여 수행할 수 있다.In another preferred embodiment of the present invention, the protein expression level measurement is a protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF ( Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, 2D electrophoresis analysis, liquid chromatography It can be performed using liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS), Western blot, and enzyme linked immunosorbent assay (ELISA).
본 발명에서는 지방증, NASH 환자를 대상으로 통합전사제 분석을 수행하여 차별 발현 유전자(Differentially Expressed Gene:DEG) 분석을 통해 선별된 유전자 및 특징 선택(Feature selection) 프로세스를 이용하여 선별된 유전자 중에서 공동으로 선별된 28종의 유전자를 최종 선별하였으며, 28종의 복합마커 또는 ATP8B1(ATPase phospholipid transporting 8B1) 마커를 이용하는 경우 만성 간질환 진단능이 현저하게 향상된 것을 확인하였으므로, 본 발명의 복합마커는 만성 간질환 진단을 위한 바이오마커로 적용할 수 있는 것을 확인하였다.In the present invention, integrated transcriptase analysis is performed on steatosis and NASH patients to jointly select genes selected through differentially expressed gene (DEG) analysis and genes selected using a feature selection process. The 28 selected genes were finally selected, and it was confirmed that the diagnostic ability of chronic liver disease was significantly improved when 28 types of complex markers or ATP8B1 (ATPase phospholipid transporting 8B1) markers were used. It was confirmed that it can be applied as a biomarker for
도 1은 만성 간질환 진단을 위한 복합마커 선별 과정을 나타낸 모식도이다.
도 2는 통합전사체 분석 후, 차별 발현 유전자(Differentially Expressed Gene:DEG) 분석을 수행한 것으로, 지방증과 NASH 환자 그룹의 유전자 발현패턴을 분석한 히트맵 및 유전자 선별과정을 나타낸 도면이다.
도 3은 마커 유전자 선별을 위한 분석 과정을 나타낸 모식도이다.
도 4는 모델링에 따라 선정된 특징 사례 및 특징 사례 선별 과정을 나타낸 도면이다.
도 5는 차별 발현 유전자(DEG) 분석을 통해 선별된 1330개 유전자 및 특징사례 분석을 통해 선별된 유전자들 중에서 공통으로 발현된 유전자를 선별 과정을 나타낸 모식도로, 총 28종의 유전자를 선별하였다.
도 6은 복합마커로 선별된 28종 유전자의 간질환 단계별 발현 패턴을 나타낸 도면이다.
도 7은 본 발명의 28종 복합마커를 이용하여 만성 간질환을 진단하였을 때, 진단의 정확성을 ROC 곡선을 통해 나타낸 도면이다.1 is a schematic diagram showing a complex marker selection process for diagnosing chronic liver disease.
2 is a diagram showing a heat map and gene selection process analyzing gene expression patterns of a group of steatosis and NASH patients by performing differentially expressed gene (DEG) analysis after integrated transcriptome analysis.
3 is a schematic diagram showing an analysis process for marker gene selection.
4 is a diagram illustrating a feature case selected according to modeling and a feature case selection process.
5 is a schematic diagram showing the selection process for genes commonly expressed among 1330 genes selected through differential expression gene (DEG) analysis and genes selected through feature case analysis, and a total of 28 genes were selected.
6 is a view showing the liver disease step-by-step expression patterns of 28 genes selected as complex markers.
7 is a diagram showing the accuracy of diagnosis through an ROC curve when chronic liver disease is diagnosed using the 28 composite markers of the present invention.
이하, 본 발명을 상세하게 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 일관점에서, AJUBA(ajuba LIM protein; NCBI Gene ID: 84962), ANXA2(annexin A2; NCBI Gene ID: 302), ATP8B1(ATPase phospholipid transporting 8B1; NCBI Gene ID: 5205), CCND1(cyclin D1; NCBI Gene ID: 595), CEBPD(CCAAT enhancer binding protein; NCBI Gene ID: 1052), CHST9(carbohydrate sulfotransferase 9; NCBI Gene ID: 83539), CLDN7(claudin 7; NCBI Gene ID: 1366), DNAJC12(DnaJ heat shock protein family (Hsp40) member C12; NCBI Gene ID: 56521), DPYSL2(dihydropyrimidinase like 2; NCBI Gene ID: 1808), FAT1(FAT atypical cadherin 1; NCBI Gene ID: 2195), GPNMB(glycoprotein nmb; NCBI Gene ID: 10457), ITGA6(integrin subunit alpha 6; NCBI Gene ID: 3655), KRT8(keratin 8; NCBI Gene ID: 3856), LGALS3(galectin 3; NCBI Gene ID: 3958), MCM3(minichromosome maintenance complex component 3; NCBI Gene ID: 4172), MCM4(minichromosome maintenance complex component 4; NCBI Gene ID: 4173), MCM6(minichromosome maintenance complex component 6; NCBI Gene ID: 4175), MSN(moesin; NCBI Gene ID: 4478), NR0B2(nuclear receptor subfamily 0 group B member 2; NCBI Gene ID: 8431), PACSIN3(protein kinase C and casein kinase substrate in neurons 3; NCBI Gene ID: 29763), PDGFRA(platelet derived growth factor receptor alpha; NCBI Gene ID: 5156), PIK3IP1(phosphoinositide-3-kinase interacting protein 1; NCBI Gene ID: 113791), RNASE1(ribonuclease A family member 1, pancreatic; NCBI Gene ID: 6035), RNF152(ring finger protein 152; NCBI Gene ID: 220441), SFXN2(sideroflexin 2; NCBI Gene ID: 118980), SULF2(sulfatase 2; NCBI Gene ID: 55959), TTC36(tetratricopeptide repeat domain 36; NCBI Gene ID: 143941) 및 WIPI1(WD repeat domain, phosphoinositide interacting 1; NCBI Gene ID: 55062)을 포함하는 복합마커; 또는 The present invention is from one point of view, AJUBA (ajuba LIM protein; NCBI Gene ID: 84962), ANXA2 (annexin A2; NCBI Gene ID: 302), ATP8B1 (ATPase phospholipid transporting 8B1; NCBI Gene ID: 5205), CCND1 (cyclin D1) ; NCBI Gene ID: 595), CEBPD (CCAAT enhancer binding protein; NCBI Gene ID: 1052), CHST9 (
ATP8B1(ATPase phospholipid transporting 8B1) 마커를 포함하는 만성 간질환 진단 마커 조성물을 제공한다.Provided is a chronic liver disease diagnostic marker composition comprising an ATPase phospholipid transporting 8B1 (ATP8B1) marker.
본 발명에서 사용된 용어 "진단"은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 진단은 만성 간질환, 특히, 지방증(Steatosis), 비-알코올성 지방간질환(non-alcoholic fatty liver disease; NAFLD), 비-알코올성 지방간염((non-alcoholic steatohepatitis; NASH) 또는 간경화증 여부를 확인하는 것이다.As used herein, the term “diagnosis” refers to ascertaining the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is chronic liver disease, in particular Steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) or To check for liver cirrhosis.
본 발명에서 사용된 용어 "진단용 바이오마커"란 정상 대조군에 비해 만성 간질환이 진행된 개체에 비해, 특정 유전자 발현 수준 또는 단백질 발현 수준의 유의적인 증가 또는 감소 양상을 보이는 폴리펩티드 또는 핵산(예: mRNA 등), 지질, 당지질, 당단백질, 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자 등을 포함하며, 바람직하게는 ATP8B1 마커 또는 상기 28종의 복합마커이다.As used herein, the term "diagnostic biomarker" refers to a polypeptide or nucleic acid (eg, mRNA, etc.) that shows a significant increase or decrease in specific gene expression level or protein expression level compared to an individual with advanced chronic liver disease compared to a normal control. ), lipids, glycolipids, glycoproteins, and organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.), and the like, preferably ATP8B1 marker or the 28 types of complex markers.
본 발명의 구체적인 일구현예에서, 도 1에 나타난 모식도와 같은 방법으로 만성 간질환 진단을 위한 마커를 선별하였다. 지방증(Steatosis), 비-알코올성 지방간염인 NASH 환자를 대상으로 통합전사제 분석을 수행하여 차별 발현 유전자(Differentially Expressed Gene:DEG) 분석을 통해 선별된 유전자(도 2)와 생명정보학 분석의 특징 선택(Feature selection) 프로세스를 이용하여 선별된 유전자(도 3 및 도 4) 중에서 공통으로 나온 28종의 유전자를 최종 선별하였다 (도 5).In a specific embodiment of the present invention, markers for diagnosing chronic liver disease were selected in the same manner as in the schematic diagram shown in FIG. 1 . Integrative transcriptase analysis was performed on NASH patients with steatosis and non-alcoholic steatohepatitis. Genes selected through differentially expressed gene (DEG) analysis (FIG. 2) and bioinformatics analysis feature selection (Feature selection) Among the selected genes ( FIGS. 3 and 4 ), 28 common genes were finally selected ( FIG. 5 ).
또한, 선별된 28종의 유전자를 지방증과 NASH 단계별로 나누어 유전자 발현 패턴을 확인한 결과(도 6), 각 단계별로 서로 상이한 패턴을 보이는 것으로 확인되었으므로, 이들의 조합을 통해 만성 간질환 각 단계별 진단이 가능하다고 판단되었다.In addition, as a result of checking the gene expression patterns by dividing the selected 28 genes into steatosis and NASH stages (FIG. 6), it was confirmed that different patterns were shown at each stage, so the diagnosis of each stage of chronic liver disease through a combination of these was confirmed. was judged possible.
본 발명의 구체적인 다른 일구현예에서, 본 발명에서 선별한 28종의 복합마커를 이용하여 만성 간질환에 대한 진단능을 평가한 결과, 도 7에 나타난 바와 같이, AUC 값은 모두 1로 통계적으로 매우 유의한 만성 간질환 진단능을 보이는 것을 확인하였다. In another specific embodiment of the present invention, as a result of evaluating the diagnostic ability for chronic liver disease using 28 types of complex markers selected in the present invention, as shown in FIG. 7 , all AUC values are statistically 1. It was confirmed that a very significant diagnostic ability for chronic liver disease was shown.
ROC 곡선은 진단법의 정확성을 비교하는 방법으로 널리 사용되는 방법 중 하나이다 (Akobeong, 2007). X축에는 `민감도', Y축에는 `1-특이도' 로 하여 그래프를 그리고, 이때 대각선을 기준으로 곡선 아래 면적이 0.5이며, 대각선보다 ROC 곡선이 위에 있을 경우 좋은 모델이라 예측하게 된다. AUC는 0.5에서 1 사이 값을 갖게 되며, 1에 가까워질수록 곡선도 넓어지고 예측한 모델이 좋다는 것을 의미한다.The ROC curve is one of the widely used methods to compare the accuracy of diagnostic methods (Akobeong, 2007). Draw a graph with ‘sensitivity’ on the X-axis and ‘1-specificity’ on the Y-axis. AUC has a value between 0.5 and 1, and the closer to 1, the wider the curve and the better the predicted model.
또한, 선별한 28종의 복합마커 중에서 일부에 대해, qPCR을 이용하여 검증(validation)을 수행한 결과, ATP8B1, CEBPD, GPNMB, KRT8 및 RNASE1 마커를 포함하는 일부 마커들이 통계적으로 유의한 결과를 보여주는 것을 확인하였다 (표 2). 즉, 본 발명의 만성 간질환 진단을 위한 ATP8B1 마커 또는 복합마커는, 만성 간질환 진단에 가장 적합한 마커인 것을 확인하였다.In addition, as a result of performing validation using qPCR for some of the selected 28 types of complex markers, some markers including ATP8B1, CEBPD, GPNMB, KRT8 and RNASE1 markers showed statistically significant results was confirmed (Table 2). That is, it was confirmed that the ATP8B1 marker or complex marker for diagnosing chronic liver disease of the present invention is the most suitable marker for diagnosing chronic liver disease.
따라서, 본 발명은 다른 관점에서, 상기 ATP8B1 마커 또는 복합마커의 mRNA 또는 단백질 수준을 측정하는 제제를 포함하는 만성 간질환 진단용 조성물에 관한 것이다.Accordingly, in another aspect, the present invention relates to a composition for diagnosing chronic liver disease comprising an agent for measuring the mRNA or protein level of the ATP8B1 marker or the complex marker.
본 발명에 있어서, 상기 만성 간질환은 지방증, 비-알코올성 지방간질환(non-alcoholic fatty liver disease; NAFLD), 비-알코올성 지방간염((non-alcoholic steatohepatitis; NASH) 또는 간경화증일 수 있다.In the present invention, the chronic liver disease may be steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) or liver cirrhosis.
본 발명에 사용된 용어 "mRNA 발현수준 측정"이란 생물학적 시료에서 만성 간질환 진단을 위한 바이오마커의 mRNA 존재 여부와 발현 정도를 확인하는 과정으로 mRNA의 양을 측정한다. 이를 위한 분석 방법으로는 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(Northern blotting), DNA 칩 등이 있으나 이로 제한되는 것은 아니다.As used herein, the term “mRNA expression level measurement” refers to a process of determining the presence and expression level of mRNA of a biomarker for diagnosing chronic liver disease in a biological sample, and measuring the amount of mRNA. Analysis methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection) assay), Northern blotting, and a DNA chip, but is not limited thereto.
상기 ATP8B1 마커 또는 복합마커의 mRNA 수준을 측정하는 제제는 상기 마커의 유전자에 특이적으로 결합하는 프라이머쌍, 프로브 또는 안티센스 뉴클레오타이드인 것을 특징으로 하며, 상기 유전자들의 핵산 정보가 GeneBank 등에 알려져 있으므로 당업자는 상기 서열을 바탕으로 이들 프라이머쌍, 프로브 또는 안티센스 뉴클레오타이드를 디자인할 수 있다.The agent for measuring the mRNA level of the ATP8B1 marker or complex marker is characterized in that it is a primer pair, probe, or antisense nucleotide that specifically binds to the gene of the marker. Based on the sequence, these primer pairs, probes or antisense nucleotides can be designed.
본 발명에서 사용된 용어 "프라이머"는 표적 유전자 서열을 인지하는 단편으로서, 정방향 및 역방향의 프라이머 쌍을 포함하나, 바람직하게는, 특이성 및 민감성을 가지는 분석 결과를 제공하는 프라이머 쌍이다.As used herein, the term "primer" refers to a fragment recognizing a target gene sequence, including a pair of forward and reverse primers, but preferably a primer pair that provides analysis results with specificity and sensitivity.
본 발명에서 사용된 용어 "프로브"란 시료 내의 검출하고자 하는 표적 물질과 특이적으로 결합할 수 있는 물질을 의미하며, 상기 결합을 통하여 특이적으로 시료 내의 표적 물질의 존재를 확인할 수 있는 물질을 의미한다. 프로브의 종류는 당업계에서 통상적으로 사용되는 물질로서 제한은 없으나, 바람직하게는 PNA(peptide nucleic acid), LNA(locked nucleic acid), 펩타이드, 폴리펩타이드, 단백질, RNA 또는 DNA 일 수 있으며, 가장 바람직하게는 PNA이다. As used herein, the term “probe” refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in a sample through the binding. do. The type of probe is not limited as a material commonly used in the art, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably It is PNA.
본 발명에서 사용된 용어 "안티센스"는 안티센스 올리고머가 왓슨-크릭 염기쌍 형성에 의해 RNA 내의 표적 서열과 혼성화되어, 표적서열 내에서 전형적으로 mRNA와 RNA:올리고머 헤테로이중체의 형성을 허용하는 뉴클레오티드 염기의 서열 및 서브유닛간 백본을 갖는 올리고머를 의미한다. 올리고머는 표적 서열에 대한 정확한 서열 상보성 또는 근사 상보성을 가질 수 있다.As used herein, the term "antisense" means that an antisense oligomer hybridizes with a target sequence in RNA by Watson-Crick base pairing, and typically mRNA and RNA: oligomeric heteroduplex formation in the target sequence. It refers to an oligomer having a sequence and an inter-subunit backbone. An oligomer may have exact sequence complementarity or approximate complementarity to a target sequence.
본 발명에 사용된 용어 "단백질 발현수준 측정"이란 생물학적 시료에서 만성 간질환 진단을 위한 바이오마커로부터 발현된 단백질의 존재 여부와 발현 정도를 확인하는 과정이다. 상기 유전자의 단백질 또는 펩타이드 단편에 특이적으로 결합하는 항체, 상호작용 단백질, 리간드, 나노입자(nanoparticles) 또는 압타머(aptamer)를 이용하여 단백질의 양을 확인할 수 있다.As used herein, the term "measurement of protein expression level" is a process of confirming the presence and expression level of a protein expressed from a biomarker for diagnosing chronic liver disease in a biological sample. The amount of the protein can be confirmed using an antibody, an interacting protein, a ligand, nanoparticles, or an aptamer that specifically binds to a protein or peptide fragment of the gene.
상기 단백질 발현 수준 측정 또는 비교 분석 방법으로는 단백질 칩 분석, 면역측정법, 리간드 바인딩 어세이, MALDI-TOF(Matrix Desorption/Ionization Time of Flight Mass Spectrometry)분석, SELDI-TOF(Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry)분석, 방사선 면역분석, 방사 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 보체 고정 분석법, 2차원 전기영동 분석, 액상 크로마토그래피-질량분석(liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS(liquid chromatography-Mass Spectrometry/ Mass Spectrometry), 웨스턴 블랏 및 ELISA(enzyme linked immunosorbent assay)등이 있으나 이로 제한되는 것은 아니다. The protein expression level measurement or comparative analysis method includes protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time) analysis. of Flight Mass Spectrometry analysis, radioimmunoassay, radioimmunodiffusion method, Ouchteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (liquid chromatography) -Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry), Western blot, ELISA (enzyme linked immunosorbent assay), etc., but are not limited thereto.
본 발명에 사용된 용어 "항체"는 항원과 특이적으로 결합하여 항원-항체 반응을 일으키는 물질을 가리킨다. 본 발명의 목적상, 항체는 본 발명의 바이오마커에 대해 특이적으로 결합하는 항체를 의미한다. 본 발명의 항체는 다클론 항체, 단클론 항체 및 재조합 항체를 모두 포함한다.As used herein, the term “antibody” refers to a substance that specifically binds to an antigen and induces an antigen-antibody reaction. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a biomarker of the present invention. Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
본 발명은 또 다른 관점에서, 상기 만성 간질환 진단용 조성물을 포함하는 만성 간질환 진단용 키트에 관한 것이다.In another aspect, the present invention relates to a kit for diagnosing chronic liver disease comprising the composition for diagnosing chronic liver disease.
상기 키트는 당업계에 알려져 있는 통상의 제조방법에 의해 제조될 수 있다. 상기 키트는 예를 들면, 동결 건조 형태의 항체와 완충액, 안정화제, 불활성 단백질 등을 포함할 수 있다. The kit may be prepared by a conventional manufacturing method known in the art. The kit may include, for example, a freeze-dried antibody, a buffer, a stabilizer, an inactive protein, and the like.
상기 키트는 검출 가능한 표지를 더 포함할 수 있다. 용어 "검출 가능한 표지"는 표지가 없는 동일한 종류의 분자들 중에서 표지를 포함하는 분자를 특이적으로 검출하도록 하는 원자 또는 분자를 의미한다. 상기 검출 가능한 표지는 상기 단백질 또는 그의 단편에 특이적으로 결합하는 항체, 상호작용 단백질, 리간드, 나노입자, 또는 압타머에 부착된 것일 수 있다. 상기 검출 가능한 표지는 방사종(radionuclide), 형광원(fluorophore), 효소(enzyme)를 포함할 수 있다. The kit may further include a detectable label. The term "detectable label" refers to an atom or molecule that allows for specific detection of a molecule comprising a label among molecules of the same type without a label. The detectable label may be attached to an antibody that specifically binds to the protein or fragment thereof, an interacting protein, a ligand, a nanoparticle, or an aptamer. The detectable label may include a radioactive species (radionuclide), a fluorescence source (fluorophore), and an enzyme (enzyme).
상기 키트는 당업계에 알려진 다양한 면역분석법 또는 면역염색법에 따라 이용될 수 있다. 상기 면역분석법 또는 면역염색법은 방사능면역분석, 방사능면역침전, 면역침전, ELISA, 캡처-ELISA, 억제 또는 경쟁 분석, 샌드위치 분석, 유세포 분석, 면역형광염색 및 면역친화성 정제를 포함할 수 있다. 바람직하게, 상기 키트는 RT-PCR(reverse transcription polymerase chain reaction) 키트, DNA 칩 키트, ELISA(enzyme linked immunosorbent assay) 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(multiple reaction monitoring)인 것일 수 있다. The kit may be used according to various immunoassays or immunostaining methods known in the art. The immunoassay or immunostaining method may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining and immunoaffinity purification. Preferably, the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or multiple reaction monitoring (MRM). have.
본 발명은 또 다른 관점에서, (a) 환자의 생물학적 시료로부터 상기 ATP8B1 마커 또는 복합마커의 mRNA 또는 단백질 발현 수준을 측정하는 단계; 및 In another aspect, the present invention, (a) measuring the mRNA or protein expression level of the ATP8B1 marker or complex marker from a biological sample of a patient; and
(b) 상기 mRNA 또는 단백질 발현 수준을 대조군 시료와 비교하는 단계를 포함하는 만성 간질환 진단을 위한 정보 제공방법에 관한 것이다.(b) relates to a method for providing information for diagnosing chronic liver disease, comprising comparing the mRNA or protein expression level with a control sample.
상기 방법에서 "생물학적 시료(biological sample)"란 조직, 세포, 혈액, 혈청, 혈장, 타액, 뇌척수액 또는 뇨와 같은 시료 등을 의미하며, 바람직하게는 혈액, 혈장, 혈청을 의미한다.In the above method, "biological sample" means a sample such as tissue, cells, blood, serum, plasma, saliva, cerebrospinal fluid or urine, and preferably means blood, plasma, or serum.
상기 만성 간질환 진단에 대한 정보 제공방법에서 ATP8B1 마커 또는 복합마커의 mRNA 또는 이의 단백질의 발현 수준 측정 방법은 상술한 바와 같다.In the method for providing information on the diagnosis of chronic liver disease, the method for measuring the expression level of the mRNA of the ATP8B1 marker or the complex marker or the protein thereof is as described above.
본 발명의 복합마커를 이용하여 만성 간질환 진단을 위한 정보를 제공하는 경우, 상기 만성 간질환 진단을 위한 정보 제공방법은 CEBPD(CCAAT enhancer binding protein), DNAJC12(DnaJ heat shock protein family (Hsp40) member C12), NR0B2(nuclear receptor subfamily 0 group B member 2), PACSIN3(protein kinase C and casein kinase substrate in neurons 3), RNF152(ring finger protein 152) 및 SFXN2(sideroflexin 2), TTC36(tetratricopeptide repeat domain 36) 마커의 mRNA 또는 이의 단백질 발현 수준이 대조군에 비해 감소하고,When providing information for diagnosing chronic liver disease using the complex marker of the present invention, the information providing method for diagnosing chronic liver disease is CEBPD (CCAAT enhancer binding protein), DNAJC12 (DnaJ heat shock protein family (Hsp40) member C12), NR0B2 (
AJUBA(ajuba LIM protein), ANXA2(annexin A2), ATP8B1(ATPase phospholipid transporting 8B1), CCND1(cyclin D1), CHST9(carbohydrate sulfotransferase 9), CLDN7(claudin 7), DPYSL2(dihydropyrimidinase like 2), FAT1(FAT atypical cadherin 1), GPNMB(glycoprotein nmb), ITGA6(integrin subunit alpha 6), KRT8(keratin 8), LGALS3(galectin 3), MCM3(minichromosome maintenance complex component 3), MCM4(minichromosome maintenance complex component 4), MCM6(minichromosome maintenance complex component 6), MSN(moesin), PDGFRA(platelet derived growth factor receptor alpha), PIK3IP1(phosphoinositide-3-kinase interacting protein 1), RNASE1(ribonuclease A family member 1, pancreatic), SULF2(sulfatase 2), 및 WIPI1(WD repeat domain, phosphoinositide interacting 1) 마커의 mRNA 또는 이의 단백질 발현 수준이 대조군에 비해 증가하면, 만성 간질환인 것으로 정보를 제공하는 단계를 추가로 포함할 수 있다.AJUBA (ajuba LIM protein), ANXA2 (annexin A2), ATP8B1 (ATPase phospholipid transporting 8B1), CCND1 (cyclin D1), CHST9 (carbohydrate sulfotransferase 9), CLDN7 (claudin 7), DPYSL2 (dihydropyrimidinase like 2), FAT1 (FAT1) atypical cadherin 1), GPNMB (glycoprotein nmb), ITGA6 (integrin subunit alpha 6), KRT8 (keratin 8), LGALS3 (galectin 3), MCM3 (minichromosome maintenance complex component 3), MCM4 (minichromosome maintenance complex component 4), MCM6 (minichromosome maintenance complex component 6), MSN (moesin), PDGFRA (platelet derived growth factor receptor alpha), PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), RNASE1 (ribonuclease
본 발명의 ATP8B1 마커를 이용하여 만성 간질환 진단을 위한 정보를 제공하는 경우, 상기 만성 간질환 진단을 위한 정보 제공방법은 ATP8B1(ATPase phospholipid transporting 8B1) 마커의 mRNA 또는 이의 단백질 발현 수준이 대조군에 비해 증가하면, 만성 간질환인 것으로 정보를 제공하는 단계를 추가로 포함할 수 있다.When providing information for diagnosing chronic liver disease using the ATP8B1 marker of the present invention, the method for providing information for diagnosing chronic liver disease is that the mRNA or protein expression level of the ATP8B1 (ATPase phospholipid transporting 8B1) marker is higher than that of the control group. If increased, it may further comprise providing information as having chronic liver disease.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
환자 선정Patient selection
본 발명에서는 만성 간질환 진단을 위한 마커를 선별하기 위해, 지방증(Steatosis) 환자 42명, 비-알코올성 지방간염인 NASH 환자 56명을 선별하여 Biopsy 또는 수술 후 시료로부터 환자의 조직을 채취하였다. In the present invention, in order to select markers for diagnosing chronic liver disease, 42 patients with steatosis and 56 patients with NASH with non-alcoholic steatohepatitis were selected, and patient tissues were collected from biopsy or postoperative samples.
차별 발현 유전자(Differentially Expressed Gene:DEG) 분석Differentially Expressed Gene (DEG) Analysis
실시예 1에서 수득한 환자의 조직을 이용하여 트리아졸(Trizol)로 RNA를 분리/정제한 후 나노드랍(nanodrop)과 피코그린(picogreen)을 이용하여 정량분석하고 애질런트(Agilent)사의 Bioanalyzer를 이용하여 정성분석을 수행했다. 이후 실험에 적합한 양과 품질의 샘플로 일루미나(Illumina)사의 TruSeq Stranded Total RNA LT Sample Prep Kit (Gold)를 사용하여 라이브러리를 구축하고 Novaseq 6000을 사용하여 염기서열분석(sequencing)을 수행하였다. 이로부터 도출된 FASTQ 파일로 다음과 같이 생명정보분석을 수행하였다. After separation/purification of RNA with triazole using the patient's tissue obtained in Example 1, quantitative analysis was performed using nanodrop and picogreen, and Agilent's Bioanalyzer was used. Thus, a qualitative analysis was performed. Afterwards, a library was constructed using Illumina's TruSeq Stranded Total RNA LT Sample Prep Kit (Gold) with samples of suitable quantity and quality for the experiment, and sequencing was performed using Novaseq 6000. Bioinformation analysis was performed as follows with the FASTQ file derived therefrom.
총 RNA 서열분석 로우 데이타(total RNA-seq raw data(.fastq))의 품질을 확인하고 이를 향상시키기 위해 Trim Galore를 이용하여 결과를 획득하였다. 이 과정에서 내부적으로 FastQC로 리드(read)의 품질을 확인하고 컷어댑트(Cutadapt)로 낮은 품질의 리드(read)나 남은 어댑터 시퀀스(adapter seq)를 제거하였다. 이후 리드(read)를 레퍼런스(reference)인 휴먼 지놈(genome)에 얼라인먼트(alignment) 하기 위해 STAR alignment tool을 이용하였다. 추후 전사체 발현량 분석을 통한 예후 예측 마커 발굴의 정확도를 높이기 위해 레퍼런스(reference)에 얼라인먼트(alignment)된 결과 파일(.bam)은 Picard mark duplication tool을 이용하여 중복(duplication)이 제거된 결과 파일(.bam)로 획득하였다. 이후, 질병의 진행 단계별 유전자 발현량을 계산하기 위해, 환자들을 지방증(Steatosis)과 NASH로 그룹핑하고 커프티프(cuffdiff)를 진행하여 유전자 수준에서 그룹별, 시료별 발현 수준을 비교 분석할 수 있도록 정규화된(normalized) 결과를 획득하였다.To check and improve the quality of total RNA-seq raw data (.fastq), the results were obtained using Trim Galore. In this process, read quality was checked internally with FastQC, and low-quality reads or remaining adapter seq were removed with Cutadapt. Thereafter, the STAR alignment tool was used to align the read with the human genome as a reference. In order to increase the accuracy of prognostic marker discovery through transcript expression level analysis later, the result file (.bam) aligned with the reference is the result file from which duplication is removed using the Picard mark duplication tool. (.bam) was obtained. Then, in order to calculate the gene expression level at each stage of the disease, the patients are grouped into steatosis and NASH and cuffdiff is performed to normalize the expression level for each group and sample at the gene level to compare and analyze. Normalized results were obtained.
또한, 통합전사체 분석을 통해 수집된 데이터를 도 2에 나타난 바와 같이, DEG (Differentially expressed gene)의 발현 차이에 따라 히트맵으로 시각화하였으며, (1) 발현 레벨(Expression Level (FPKM)) 컷오프 > 1, (2) p-value < 0.05, (3) Log2 Fold Change > |1.3| 및 (4) Standard Deviation < Mean 조건에 맞는 유전자 1330 개를 선별하였다.In addition, as shown in Figure 2, the data collected through the integrated transcriptome analysis was visualized as a heat map according to the difference in the expression of differentially expressed gene (DEG), and (1) Expression Level (FPKM) cutoff > 1, (2) p-value < 0.05, (3) Log2 Fold Change > |1.3| and (4) 1330 genes satisfying the Standard Deviation < Mean condition were selected.
특징 선택 (Feature selection)에 의한 유전자 선별Gene selection by feature selection
유전자 선별을 위해서는 도 4에 기재된 특징 선택(Feature selection) 프로세스와 특징중요도(Feature importance) 방식의 분석을 수행하였으며, 도 3에 기재된 워크플로우(WorkFlow)와 같이 진행하였다.For gene selection, the feature selection process and feature importance method described in FIG. 4 were analyzed, and the workflow described in FIG. 3 was performed.
특징 선택(Feature selection) 방식에서는 파이썬(python)으로 개발된 selectKBest 방법을 이용하여 반복적으로 유전자를 제거하면서 선형 지지 벡터 머신(linear Support Vector Machine) 모델에 의해 잘 분류되는 유전자 세트를 선택하였다 (참조 : https://scikit-learn.org/). 유의미한 유전자 그룹 평가에 있어서 AUC(area under the curve)와 ROC(Receiver operating characteristic) ACC(accuracy) 분석을 수행한 바, AUC의 경우에는 정확도(accuracy) 방식으로 계산한 후 ANOVA를 이용하여 통계적으로 두 그룹의 차이가 있는 지를 확인하고 이를 통해 628개의 유전자를 선별하였고, ROC ACC의 경우에는 ROC AUC 방식으로 계산한 후 ANOVA(Analysis of variance)를 이용하여 통계적으로 두 그룹의 차이가 있는 지 확인하고 147개의 적합한 유전자를 선별하였다. In the feature selection method, a gene set that is well classified by the linear support vector machine model was selected while repeatedly removing genes using the selectKBest method developed in Python (see: https://scikit-learn.org/). In the evaluation of significant gene groups, AUC (area under the curve) and ROC (Receiver operating characteristic) ACC (accuracy) analysis was performed. In the case of AUC, after calculating in the accuracy method, statistically two After checking whether there is a difference between the groups, 628 genes were selected through this. In the case of ROC ACC, after calculating using the ROC AUC method, analysis of variance (ANOVA) was used to determine whether there was a statistically significant difference between the two
또한 특징중요도(Feature importance) 방식에서는, 파이썬으로 개발된 lightGBM 방법(unsupervised machine learning)을 이용하여 회귀 모델(regression model) 기법으로 계산하였을 때, 두 그룹을 분류하는데 있어 가장 잘 분류된다고 계산되는 순서대로 100개의 유전자를 선별하였다 (참조 : https://lightgbm.readthedocs.io/en/latest/).In addition, in the feature importance method, when calculated using the regression model method using the lightGBM method developed in Python (unsupervised machine learning), the two groups are classified in the order in which they are calculated best. 100 genes were selected (see: https://lightgbm.readthedocs.io/en/latest/).
복합마커 선별 및 만성 간질환 단계별 발현 패턴 분석Complex marker selection and chronic liver disease step-by-step expression pattern analysis
본 발명에서는 도 5에 나타낸 모식도와 같이, 차별 발현 유전자(Differentially Expressed Gene:DEG) 분석을 통해 선별된 유전자와 특징 선택(Feature selection) 프로세스를 이용하여 선별된 유전자 중에서 공통으로 선별된 28종의 유전자를 최종 선별하였다.In the present invention, as shown in the schematic diagram shown in FIG. 5 , 28 genes selected in common among genes selected through differentially expressed gene (DEG) analysis and genes selected using a feature selection process was finally selected.
또한, 선별된 28종의 유전자를 지방증(Steatosis) 단계와 NASH 단계로 나누어 유전자 발현 패턴을 확인한 결과, 도 6 및 표 3에 나타난 바와 같이, 각 단계별로 서로 상이한 패턴을 보이는 것으로 확인되었으므로, 이들의 조합을 통해 만성 간질환 각 단계별 진단이 가능한 것을 확인하였다.In addition, as a result of confirming gene expression patterns by dividing the selected 28 genes into a steatosis stage and a NASH stage, as shown in FIG. 6 and Table 3, it was confirmed that different patterns were shown at each stage, so their Through the combination, it was confirmed that the diagnosis of each stage of chronic liver disease was possible.
해당 유전자들은 지방증(Steatosis)에서 NASH로 진행됨에 따라 유전자의 발현량이 감소하는 7개의 유전자 CEBPD, DNAJC12, NR0B2, PACSIN3, RNF152, SFXN2, TTC36 와 발현이 증가하는 21개의 유전자 AJUBA, ANXA2, ATP8B1, CCND1, CHST9, CLDN7, DPYSL2, FAT1, GPNMB, ITGA6, KRT8, LGALS3, MCM3, MCM4, MCM6, MSN, PDGFRA, PIK3IP1, RNASE1, SULF2, WIPI1 들이다.The corresponding genes are seven genes CEBPD, DNAJC12, NR0B2, PACSIN3, RNF152, SFXN2, TTC36, whose expression levels decrease with the progression from steatosis to NASH, and 21 genes AJUBA, ANXA2, ATP8B1, CCND1 whose expression increases. , CHST9, CLDN7, DPYSL2, FAT1, GPNMB, ITGA6, KRT8, LGALS3, MCM3, MCM4, MCM6, MSN, PDGFRA, PIK3IP1, RNASE1, SULF2, WIPI1.
복합마커의 만성 간질환 진단능 확인Confirmation of chronic liver disease diagnostic ability of complex markers
본 발명에서는 선별한 28종의 복합마커를 이용하여 만성 간질환에 대한 진단능을 평가하기 위해, 상기 실시예 1의 비-알코올성 지방간염인 NASH 환자를 대상으로 ROC 곡선 분석을 수행하여 진단의 정확도를 분석하였다.In the present invention, in order to evaluate the diagnostic ability for chronic liver disease using 28 selected complex markers, the ROC curve analysis was performed on NASH patients with non-alcoholic steatohepatitis of Example 1, and the accuracy of diagnosis was analyzed.
ROC 곡선은 결과의 가양성률(FPR, 1-specificity)과 진양성률(TPR, sensitivity)을 나타내며, 검사의 정확도를 평가한다. 28종의 복합마커를 이용하여 지방간과 NASH 그룹 분류의 ROC를 그려보았고, 추정방법으로는 경험적(empirical) 방법, 모수적(binormal) 방법과 비모수적(non-parametirc) 방법의 세가지를 사용하였다. The ROC curve indicates the false positive rate (FPR, 1-specificity) and the true positive rate (TPR, sensitivity) of the result, and evaluates the accuracy of the test. The ROC of fatty liver and NASH group classification was drawn using 28 complex markers, and three of the empirical, binormal, and non-parametric methods were used as estimation methods.
그 결과, 도 7에 나타난 바와 같이, AUC 값은 모두 1로 매우 정확한 만성 간질환 진단능을 보이는 것을 확인하였다. As a result, as shown in FIG. 7 , the AUC values were all 1, confirming that the chronic liver disease diagnosis ability was very accurate.
복합마커의 만성 간질환 진단능 검증Verification of chronic liver disease diagnostic ability of complex markers
본 발명에서는 지방증(Steatosis) 환자 및 비-알코올성 지방간염인 NASH 환자를 대상으로 하여 선별한 28종의 복합마커의 만성 간질환 진단능을 검증하였으며, 28종의 마커 중에서 일부에 대해, qPCR을 이용하여 검증(validation)을 수행하였다. In the present invention, the diagnostic ability of 28 types of complex markers selected for patients with steatosis and NASH patients with non-alcoholic steatohepatitis was verified, and for some of the 28 markers, qPCR was used. Thus, validation was performed.
실시예 2의 환자 조직으로부터 수득한 RNA를 역전사 시켜 cDNA를 제조한 다음, 하기 표 4의 프라이머 서열을 이용하여 PCR을 수행하였다. RNA obtained from the patient tissue of Example 2 was reverse transcribed to prepare cDNA, and then PCR was performed using the primer sequences shown in Table 4 below.
RNA는 10 uM 정방향 프라이머 1㎕, 10 uM 역방향 프라이머 1㎕, 증류수 1㎕, SYBR Green I(Roche사 제품) 5㎕ 및 1/5로 희석된 cDNA 2㎕를 혼합한 다음, LightCycler 96과 SYBR Green I(Roche사 제품)을 이용하여 PCR을 수행하였다. For RNA, 1 μl of 10 uM forward primer, 1 μl of 10 uM reverse primer, 1 μl of distilled water, 5 μl of SYBR Green I (Roche) and 2 μl of cDNA diluted 1/5 were mixed, followed by LightCycler 96 and SYBR Green PCR was performed using I (manufactured by Roche).
PCR은 95 ℃에서 5분 동안 반응(pre-incubation) -> 95 ℃에서 10초, 60 ℃에서 10초, 및 72 ℃에서 10초씩 45 사이클(amplification) 반복 -> 95 ℃에서 5초 -> 65 ℃에서 1분(melting curve) -> 40 ℃ 쿨링 과정으로 수행하였다.PCR reaction (pre-incubation) for 5 minutes at 95 °C -> 45 cycles (amplification) repeated at 95 °C for 10 seconds, 60 °C for 10 seconds, and 72 °C for 10 seconds -> 95 °C for 5 seconds -> 65 1 minute (melting curve) at ℃ -> 40 ℃ cooling process was performed.
PCR 완료 후, 18s rRNA값으로 정규화(normalize) 한 후 Δ2-Ct 값을 구하여 t-test를 수행하였다.After PCR was completed, t-test was performed to obtain the Δ2-Ct value after normalization to the 18s rRNA value.
Steatosis/NASHnumber of patients
Steatosis/NASH
그 결과, ATP8B1, CEBPD, GPNMB, KRT8 및 RNASE1 마커를 포함하는 일부 마커들이 통계적으로 유의한 결과를 보여주는 것을 확인하였다. 즉, 본 발명의 복합마커는 만성 간질환 진단에 적합한 것을 확인하였으며, 28종의 마커 중에서 ATP8B1, CEBPD, GPNMB, KRT8 및 RNASE1 마커는 단일마커로도 만성 간질환 진단에 활용될 수 있음을 확인하였다.As a result, it was confirmed that some markers including ATP8B1, CEBPD, GPNMB, KRT8 and RNASE1 markers showed statistically significant results. That is, it was confirmed that the complex marker of the present invention is suitable for diagnosing chronic liver disease, and among the 28 markers, ATP8B1, CEBPD, GPNMB, KRT8 and RNASE1 markers can be used for diagnosing chronic liver disease even as a single marker. .
<110> NATIONAL CANCER CENTER <120> Combined Biomarkers for Diagnosis of Chronic Hepatic Disease Based on Integrated Transcriptome Analysis and Use Thereof <130> PDPC212234k01 <150> KR 10-2020-0162459 <151> 2020-11-27 <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AJUBA_forward primer <400> 1 tctgcaggcc tggcagaaga 20 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> AJUBA_reverse primer <400> 2 cctaagctca gactgcacac atg 23 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ANXA2_forward primer <400> 3 tcggacacat ctggtgactt cc 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ANXA2_reverse primer <400> 4 cctcttcact ccagcgtcat ag 22 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ATP8B1_forward primer <400> 5 gtcttggaca gagtcacttc 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ATP8B1_reverse primer <400> 6 cgtcttatca gagaagatat aat 23 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CEBPD_forward primer <400> 7 tccggcagtt cttcaagcag ct 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CEBPD_reverse primer <400> 8 gaggtatggg tcgttgctga gt 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GPNMB_forward primer <400> 9 ctggagctga gtaggattcc tg 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GPNMB_reverse primer <400> 10 aggacgtctg tcatctggat ga 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KRT8_forward primer <400> 11 gctgaccgac gagatcaact tc 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KRT8_reverse primer <400> 12 tatcctcgta ctgtgccttg ac 22 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LGALS3_forward primer <400> 13 ccatcttctg gacagccaag tg 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LGALS3_reverse primer <400> 14 tatcagcatg cgaggcacca ct 22 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MCM6_forward primer <400> 15 gacaacagga gaagggacct ct 22 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MCM6_reverse primer <400> 16 ggacgcttta ccactggtgt ag 22 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MSN_forward primer <400> 17 caccgggaag cagctatttg a 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MSN_reverse primer <400> 18 agaacttggc acggaactta a 21 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RNASE1_forward primer <400> 19 cagtgaacac ctttgtgcac gag 23 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RNASE1_reverse primer <400> 20 tgctggagtt gctcttgtag ca 22 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA_forward primer <400> 21 cggctttggt gactctagat 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA_reverse primer <400> 22 gcgactacca tcgaaagttg 20 <110> NATIONAL CANCER CENTER <120> Combined Biomarkers for Diagnosis of Chronic Hepatic Disease Based on Integrated Transcriptome Analysis and Use Thereof <130> PDPC212234k01 <150> KR 10-2020-0162459 <151> 2020-11-27 <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AJUBA_forward primer <400> 1 tctgcaggcc tggcagaaga 20 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> AJUBA_reverse primer <400> 2 cctaagctca gactgcacac atg 23 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ANXA2_forward primer <400> 3 tcggacacat ctggtgactt cc 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ANXA2_reverse primer <400> 4 cctcttcact ccagcgtcat ag 22 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ATP8B1_forward primer <400> 5 gtcttggaca gagtcacttc 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ATP8B1_reverse primer <400> 6 cgtcttatca gagaagatat aat 23 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CEBPD_forward primer <400> 7 tccggcagtt cttcaagcag ct 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CEBPD_reverse primer <400> 8 gaggtatggg tcgttgctga gt 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GPNMB_forward primer <400> 9 ctggagctga gtaggattcc tg 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GPNMB_reverse primer <400> 10 aggacgtctg tcatctggat ga 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KRT8_forward primer <400> 11 gctgaccgac gagatcaact tc 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KRT8_reverse primer <400> 12 tatcctcgta ctgtgccttg ac 22 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LGALS3_forward primer <400> 13 ccatcttctg gacagccaag tg 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LGALS3_reverse primer <400> 14 tatcagcatg cgaggcacca ct 22 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MCM6_forward primer <400> 15 gacaacagga gaagggacct ct 22 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MCM6_reverse primer <400> 16 ggacgcttta ccactggtgt ag 22 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MSN_forward primer <400> 17 caccgggaag cagctatttg a 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MSN_reverse primer <400> 18 agaacttggc acggaactta a 21 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RNASE1_forward primer <400> 19 cagtgaacac ctttgtgcac gag 23 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RNASE1_reverse primer <400> 20 tgctggagtt gctcttgtag ca 22 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA_forward primer <400> 21 cggctttggt gactctagat 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA_reverse primer <400> 22 gcgactacca tcgaaagttg 20
Claims (10)
ATP8B1(ATPase phospholipid transporting 8B1) 마커를 포함하는 만성 간질환 진단 마커 조성물.
AJUBA (ajuba LIM protein), ANXA2 (annexin A2), ATP8B1 (ATPase phospholipid transporting 8B1), CCND1 (cyclin D1), CEBPD (CCAAT enhancer binding protein), CHST9 (carbohydrate sulfotransferase 9), CLDN7 (claudin 7), DNAJC12 ( DnaJ heat shock protein family (Hsp40) member C12), DPYSL2 (dihydropyrimidinase like 2), FAT1 (FAT atypical cadherin 1), GPNMB (glycoprotein nmb), ITGA6 (integrin subunit alpha 6), KRT8 (keratin 8), LGALS3 (galectin) 3), MCM3 (minichromosome maintenance complex component 3), MCM4 (minichromosome maintenance complex component 4), MCM6 (minichromosome maintenance complex component 6), MSN (moesin), NR0B2 (nuclear receptor subfamily 0 group B member 2), PACSIN3 (protein) kinase C and casein kinase substrate in neurons 3), PDGFRA (platelet derived growth factor receptor alpha), PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), RNASE1 (ribonuclease A family member 1, pancreatic), RNF152 (ring finger protein 152) ), SFXN2 (sideroflexin 2), SULF2 (sulfatase 2), TTC36 (tetratricopeptide repeat dom ain 36) and a complex marker including WIPI1 (WD repeat domain, phosphoinositide interacting 1); or
A chronic liver disease diagnostic marker composition comprising an ATPase phospholipid transporting 8B1 (ATP8B1) marker.
According to claim 1, wherein the chronic liver disease is steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) or liver cirrhosis, characterized in that A chronic liver disease diagnostic marker composition.
AJUBA (ajuba LIM protein), ANXA2 (annexin A2), ATP8B1 (ATPase phospholipid transporting 8B1), CCND1 (cyclin D1), CEBPD (CCAAT enhancer binding protein), CHST9 (carbohydrate sulfotransferase 9), CLDN7 (claudin 7), DNAJC12 ( DnaJ heat shock protein family (Hsp40) member C12), DPYSL2 (dihydropyrimidinase like 2), FAT1 (FAT atypical cadherin 1), GPNMB (glycoprotein nmb), ITGA6 (integrin subunit alpha 6), KRT8 (keratin 8), LGALS3 (galectin) 3), MCM3 (minichromosome maintenance complex component 3), MCM4 (minichromosome maintenance complex component 4), MCM6 (minichromosome maintenance complex component 6), MSN (moesin), NR0B2 (nuclear receptor subfamily 0 group B member 2), PACSIN3 (protein) kinase C and casein kinase substrate in neurons 3), PDGFRA (platelet derived growth factor receptor alpha), PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), RNASE1 (ribonuclease A family member 1, pancreatic), RNF152 (ring finger protein 152) ), SFXN2 (sideroflexin 2), SULF2 (sulfatase 2), TTC36 (tetratricopeptide repeat dom ain 36) and WIPI1 (WD repeat domain, phosphoinositide interacting 1), a complex marker for diagnosing chronic liver disease; Or ATP8B1 (ATPase phospholipid transporting 8B1) chronic liver disease diagnostic composition comprising an agent for measuring mRNA or protein level of a marker for diagnosing chronic liver disease.
4. The method of claim 3, wherein the chronic liver disease is steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or cirrhosis of the liver. A composition for diagnosing chronic liver disease.
The composition for diagnosing chronic liver disease according to claim 3, wherein the agent for measuring the mRNA level is a primer pair, a probe, or an antisense nucleotide that specifically binds to a marker.
The method according to claim 3, wherein the agent for measuring the protein level is an antibody, an interacting protein, a ligand, nanoparticles or an aptamer that specifically binds to a peptide fragment of a marker or a complex marker. A composition for diagnosing chronic liver disease.
A kit for diagnosing chronic liver disease, comprising the composition for diagnosing chronic liver disease according to any one of claims 3 to 6.
(b) 상기 mRNA 또는 단백질 발현 수준을 대조군 시료와 비교하는 단계를 포함하는 만성 간질환 진단을 위한 정보 제공방법.
(a) measuring the mRNA or protein expression level of the chronic liver disease diagnostic marker composition of claim 1 from the patient's biological sample; and
(B) A method for providing information for diagnosing chronic liver disease, comprising comparing the mRNA or protein expression level with a control sample.
CEBPD(CCAAT enhancer binding protein), DNAJC12(DnaJ heat shock protein family (Hsp40) member C12), NR0B2(nuclear receptor subfamily 0 group B member 2), PACSIN3(protein kinase C and casein kinase substrate in neurons 3), RNF152(ring finger protein 152) 및 SFXN2(sideroflexin 2), TTC36(tetratricopeptide repeat domain 36) 마커의 mRNA 또는 이의 단백질 발현 수준이 대조군에 비해 감소하고,
AJUBA(ajuba LIM protein), ANXA2(annexin A2), ATP8B1(ATPase phospholipid transporting 8B1), CCND1(cyclin D1), CHST9(carbohydrate sulfotransferase 9), CLDN7(claudin 7), DPYSL2(dihydropyrimidinase like 2), FAT1(FAT atypical cadherin 1), GPNMB(glycoprotein nmb), ITGA6(integrin subunit alpha 6), KRT8(keratin 8), LGALS3(galectin 3), MCM3(minichromosome maintenance complex component 3), MCM4(minichromosome maintenance complex component 4), MCM6(minichromosome maintenance complex component 6), MSN(moesin), PDGFRA(platelet derived growth factor receptor alpha), PIK3IP1(phosphoinositide-3-kinase interacting protein 1), RNASE1(ribonuclease A family member 1, pancreatic), SULF2(sulfatase 2), 및 WIPI1(WD repeat domain, phosphoinositide interacting 1) 마커의 mRNA 또는 이의 단백질 발현 수준이 대조군에 비해 증가하면, 만성 간질환인 것으로 정보를 제공하는 단계를 추가로 포함하는 것을 특징으로 하는 만성 간질환 진단을 위한 정보 제공방법.
The method of claim 8, wherein the information providing method for diagnosing chronic liver disease comprises:
CCAAT enhancer binding protein (CEBPD), DNAJC12 (DnaJ heat shock protein family (Hsp40) member C12), NR0B2 (nuclear receptor subfamily 0 group B member 2), PACSIN3 (protein kinase C and casein kinase substrate in neurons 3), RNF152 ( ring finger protein 152) and SFXN2 (sideroflexin 2), TTC36 (tetratricopeptide repeat domain 36) marker mRNA or its protein expression levels are decreased compared to the control group,
AJUBA (ajuba LIM protein), ANXA2 (annexin A2), ATP8B1 (ATPase phospholipid transporting 8B1), CCND1 (cyclin D1), CHST9 (carbohydrate sulfotransferase 9), CLDN7 (claudin 7), DPYSL2 (dihydropyrimidinase like 2), FAT1 (FAT1) atypical cadherin 1), GPNMB (glycoprotein nmb), ITGA6 (integrin subunit alpha 6), KRT8 (keratin 8), LGALS3 (galectin 3), MCM3 (minichromosome maintenance complex component 3), MCM4 (minichromosome maintenance complex component 4), MCM6 (minichromosome maintenance complex component 6), MSN (moesin), PDGFRA (platelet derived growth factor receptor alpha), PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), RNASE1 (ribonuclease A family member 1, pancreatic), SULF2 (sulfatase 2) ), and WIPI1 (WD repeat domain, phosphoinositide interacting 1) when the mRNA or protein expression level of the marker increases compared to the control, chronic liver disease, characterized in that it further comprises the step of providing information as chronic liver disease How to provide information for diagnosis.
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