KR20220148615A - 위암의 예후 예측 방법 - Google Patents
위암의 예후 예측 방법 Download PDFInfo
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- KR20220148615A KR20220148615A KR1020210055921A KR20210055921A KR20220148615A KR 20220148615 A KR20220148615 A KR 20220148615A KR 1020210055921 A KR1020210055921 A KR 1020210055921A KR 20210055921 A KR20210055921 A KR 20210055921A KR 20220148615 A KR20220148615 A KR 20220148615A
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Abstract
본 발명에 따른 조성물 및 방법을 이용하는 경우 악성 위암을 진단할 수 있으며, 위암의 예후를 예측하기 위해 매우 효과적으로 사용될 수 있다.
Description
본 발명은 악성 위암을 진단하고, 위암의 예후를 예측하는 방법에 관한 것이다.
암 (Cancer)이란 조직을 이루고 있는 세포가 비 정상적으로 무제한 증식하여 종양이 형성되도록 하고, 그에 따라 장기가 정상적인 기능을 수행할 수 없도록 하여 개체의 생명을 위협할 수 있는 매우 치명적인 질환이다. 2017 년 한국인 사망 원인 1 위가 악성 신생물(암)이었으며, 전체 사망자 중에서 27.6 %가 암으로 인해 사망하였다. 특히 위암 (gastric cancer; GC)은 세계에서 세 번째로 흔한 치명적인 암으로 인종, 성별 및 지역에 따라 암의 발생 부위에 있어서 차이가 있지만, 최근 분자 유전체 기술을 통해 분자적 특성에 따라 위암의 유형을 분류할 수 있게 되었다.
위암의 생물학적으로 관련된 하위 유형 중 특히 줄기 유사 특성을 가진 유형은 치료가 어려운 악성인 생물학적 특성을 가지며 표준 치료 화학 요법에 대한 무반응으로 인해 최악의 예후가 나타나며, 면역 체크 포인트 봉쇄 요법도 줄기 유사/EMT 암 아형에 대해 효과가 없는 것으로 확인되면서 이에 대하여 표적 치료법을 적용할 수 없는 문제점이 있다.
이에 본 발명자들은 줄기 유사 아형 GC의 분자 및 대사 특성을 연구하여 악성 위암을 효과적으로 진단할 수 있는 방법을 개발하기에 이르렀다.
본 발명의 일 목적은 위암의 예후를 진단하기 위한 조성물을 제공하는 것이다.
본 발명의 다른 목적은 위암 환자의 예후 예측을 위한 키트를 제공하는 것이다.
본 발명의 또 다른 목적은 위암 환자의 예후 예측을 위한 정보를 제공하는 방법을 제공하는 것이다.
본 발명의 또 다른 목적은 위암 환자의 예후 예측을 위한 진단기기를 제공하는 것이다.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당 업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.
이하, 본원에 기재된 다양한 구체예가 도면을 참조로 기재된다. 하기 설명에서, 본 발명의 완전한 이해를 위해서, 다양한 특이적 상세 사항, 예컨대, 특이적 형태, 조성물 및 공정 등이 기재되어 있다. 그러나, 특정의 구체예는 이들 특이적 상세 사항 중 하나 이상 없이, 또는 다른 공지된 방법 및 형태와 함께 실행될 수 있다. 다른 예에서, 공지된 공정 및 제조 기술은 본 발명을 불필요하게 모호하게 하지 않게 하기 위해서, 특정의 상세사항으로 기재되지 않는다. "한 가지 구체예" 또는 "구체예"에 대한 본 명세서 전체를 통한 참조는 구체예와 결부되어 기재된 특별한 특징, 형태, 조성 또는 특성이 본 발명의 하나 이상의 구체예에 포함됨을 의미한다. 따라서, 본 명세서 전체에 걸친 다양한 위치에서 표현된 "한 가지 구체예에서" 또는 "구체예"의 상황은 반드시 본 발명의 동일한 구체예를 나타내지는 않는다. 추가로, 특별한 특징, 형태, 조성, 또는 특성은 하나 이상의 구체예에서 어떠한 적합한 방법으로 조합될 수 있다.
명세서 내에 특별한 정의가 없으면 본 명세서에 사용된 모든 과학적 및 기술적인 용어는 본 발명이 속하는 기술분야에서 당업자에 의하여 통상적으로 이해되는 것과 동일한 의미를 가진다.
본 발명의 일 구현 예에 따르면, 암의 예후 예측용 조성물에 관한 것이다.
본 발명에서 상기 조성물은 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준을 측정하는 제제를 포함할 수 있다.
본 발명에서 상기 "트랜스젤린 (Transgelin; TAGLN)"이란 인간에서 TAGLN 유전자에 의해 코딩되는 단백질을 말한다. 이 유전자에 의해 코딩된 단백질은 섬유 아세포 및 평활근에서 발견되는 형질 전환 및 형태-변화 민감성 액틴 가교 결합/겔화 단백질로 알려져 있다. 상기 TAGLN 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 6876), TAGLN 단백질의 아미노산 서열을 서열번호 1에 나타내었으며, 본 발명에서 상기 TAGLN 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "칼데스몬 (caldesmon 1; CALD1)"이란 인간에서 CALD1 유전자에 의해 코딩되는 단백질로서 칼모듈린 결합 단백질을 말한다. 칼포닌과 마찬가지로, 칼데스몬은 평활근에서 미오신의 ATPase 활성을 억제하는 것으로도 알려져 있다. 상기 CALD1 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 800), CALD1 단백질의 아미노산 서열을 서열번호 2에 나타내었으며, 본 발명에서 상기 CALD1 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "미오신 경쇄 9 (myosin light chain 9; MYL9)"란 인간에서 MYL9 유전자에 의해 코딩되는 단백질을 말한다. 근육의 구조적 구성 요소인 미오신은 2 개의 중쇄와 4 개의 경쇄로 구성되고, 이 유전자에 의해 코딩된 단백질은 미오신 헤드의 ATPase 활성을 조절하여 근육 수축을 조절할 수 있는 미오신 경쇄인 것으로 알려져 있다. 상기 MYL9 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 10398), MYL9 단백질의 아미노산 서열을 서열번호 3에 나타내었으며, 본 발명에서 상기 MYL9 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "미오신 경쇄 키네이즈 (myosin light chain kinase; MYLK)"란 인간에서 MYLK 유전자에 의해 코딩되는 효소로서, 면역 글로불린 수퍼 패밀리의 근육 구성원인 이 유전자는 칼슘/칼모듈린 의존 효소인 미오신 경쇄 키나아제를 코딩한다. 상기 키나아제는 미오신 조절 경쇄를 인산화하여 액틴 필라멘트와 미오신 상호 작용을 촉진하여 수축 활성을 생성하는 것으로도 알려져 있다. 상기 MYLK 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 4638), MYLK 단백질의 아미노산 서열을 서열번호 4에 나타내었으며, 본 발명에서 상기 MYLK 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "액틴 알파 2 (actin alpha 2; ACTA2)"란 인간에서 ACTA2 유전자에 의해 코딩되는 단백질로서, 액틴은 마이크로 필라멘트를 형성하는 구상 다기능 단백질(globular multi-functional proteins)의 패밀리이다. ACTA2는 6 가지 액틴 이소형 중 하나이며 평활근의 수축 장치에 관여하는 것으로도 알려져 있다. 상기 ACTA2 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 59), ACTA2 단백질의 아미노산 서열을 서열번호 5에 나타내었으며, 본 발명에서 상기 ACTA2 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "트로포미오신 2 (tropomyosin 2; TPM2)"란 베타-트로포미오신 (β-tropomyosin)으로도 명하며, 인간에서 TPM2 유전자에 의해 코딩되는 단백질을 말한다. 액틴 필라멘트를 안정화시키고 근육 수축을 조절하는 기능을 하는 것으로 알려져 있다. 상기 TPM2 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 7169), TPM2 단백질의 아미노산 서열을 서열번호 6에 나타내었으며, 본 발명에서 상기 TPM2 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "칼포닌 1 (calponin 1; CNN1)"란 인간에서 CNN1 유전자에 의해 코딩되는 평활근 단백질을 말한다. 인간 염색체 게놈에서 19p13.2-p13.1에 위치하고, 액틴 필라멘트 관련 조절 단백질인 칼포닌 1을 코딩하는 7 개의 엑손을 포함하는 것으로 알려져 있다. 상기 CNN1 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 1264), CNN1 단백질의 아미노산 서열을 서열번호 7에 나타내었으며, 본 발명에서 상기 CNN1 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "모에신 (moesin; MSN)"란 인간에서 MSN 유전자에 의해 코딩되는 단백질을 말한다. 원형질 막과 액틴계 세포 골격 사이의 가교제로서 기능하는 ERM 단백질 패밀리의 구성원으로 알려져 있다. 상기 MSN 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 4478), MSN 단백질의 아미노산 서열을 서열번호 8에 나타내었으며, 본 발명에서 상기 MSN 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "칼포닌 3 (Calponin 3; CNN3)"란 인간에서 CNN3 유전자에 의해 코딩되는 단백질을 말한다. 인간 염색체 게놈에서 1p22-p21에 위치하는 것으로 알려져 있다. 상기 CNN3 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 1266), CNN3 단백질의 아미노산 서열을 서열번호 9에 나타내었으며, 본 발명에서 상기 CNN3 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "미오신 중쇄 10 (myosin heavy chain 10; MYH10)"이란 비 근육 미오신 IIB (NM-IIB)로도 명하며, 인간에서 MYH10 유전자에 의해 코딩되는 단백질을 말한다. 골격근, 심장 근육 및 평활근 미오신을 포함하는 미오신 II 서브 패밀리 단백질의 일부로 NM-IIB 및 일반적으로 비 근육 미오신은 인간의 모든 조직에서 널리 발현되는 것으로 알려져 있다. 상기 MYH10 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 4628), MYH10 단백질의 아미노산 서열을 서열번호 10에 나타내었으며, 본 발명에서 상기 MYH10 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "미오신 중쇄 11 (myosin heavy chain 11; MYH11)"이란 인간에서 MYH11 유전자에 의해 코딩되는 단백질을 말한다. 미오신 중쇄 패밀리에 속하는 평활근 미오신으로 ATP의 가수 분해를 통해 화학 에너지를 기계적 에너지로 변환하는 주요 수축 단백질인 것으로도 알려져 있다. 상기 MYH11 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 4629), MYH11 단백질의 아미노산 서열을 서열번호 11에 나타내었으며, 본 발명에서 상기 MYH11 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "시스테인이 풍부한 단백질 2(cysteine rich protein 2; CRIP2)"이란 인간에서 CRIP2 유전자에 의해 코딩되는 단백질을 말한다. 2 개의 LIM 도메인을 함유하는 단백질인 것으로도 알려져 있다. 상기 CRIP2 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 1397), CRIP2 단백질의 아미노산 서열을 서열번호 12에 나타내었으며, 본 발명에서 상기 CRIP2 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "인테그린 서브유닛 알파 5(integrin subunit alpha 5; ITGA5)"이란 인간에서 ITGA5 유전자에 의해 코딩되는 단백질을 말한다. 상기 유전자의 산물은 인테그린 알파 쇄 패밀리에 속하며, 세포 외 도메인에서 번역 후 절단을 거쳐 베타 1과 결합하여 피브로넥틴 수용체를 형성하는 이황화기가 결합한 경쇄 및 중쇄(disulfide-linked light and heavy chains)를 생성하는 것으로도 알려져 있다. 상기 ITGA5 단백질 및 유전자 정보는 NCBI(National Center for Biotechnology Information)에 등록되어 있으며(Gene ID: 3678), ITGA5 단백질의 아미노산 서열을 서열번호 13에 나타내었으며, 본 발명에서 상기 ITGA5 단백질 또는 이를 코딩하는 유전자는 인간 유래이지만, 인간에 한정하지 않고 다양한 종을 모두 포함시킬 수 있다.
본 발명에서 상기 "예후"란 질병의 경과 및 사망 또는 생존의 결과를 미리 예측하는 행위를 말한다. 상기 예후 또는 예후 진단이란 질환의 경과가 환자의 생리적 또는 환경적 상태에 따라 달라질 수 있으며, 이러한 환자의 상태를 종합적으로 고려하여 치료 전/후 질병의 경과를 예측하는 모든 행위를 의미하는 것으로 해석될 수 있다. 본 발명의 목적상 상기 예후는 암 줄기세포의 포함 여부를 예측하는 등에 따라 수행될 수 있고, 이에 따라 암의 치료 경과, 암 환자의 생존율 또는 생존 기간을 미리 예상하는 행위로 해석될 수 있다.
본 발명의 조성물에서 예후 예측의 대상은 암이 발병하였거나 발병 가능성이 높은 목적하는 개체일 수 있다. 본 발명에서 상기 "목적하는 개체"란 인간을 포함하는 포유 동물로, 예를 들면, 인간, 래트, 마우스, 모르모트, 햄스터, 토끼, 원숭이, 개, 고양이, 소, 말, 돼지, 양 및 염소로 구성된 군으로부터 선택될 수 있고, 바람직하게는 인간일 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 "인간"은 암이 발생하였거나 그 발생이 의심되는 자로, 암의 적절한 치료가 필요하거나 예상되는 환자를 의미하는 것일 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 예후 예측의 대상이 되는 질환으로 상기 "암"은 포유류에서 전형적으로 조절되지 않는 세포 성장으로 특징 지어진 생리적 상태를 나타내거나 가리킨다. 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종일 수 있으나, 종양의 분화 및/또는 증식 등 암의 진행이 본 발명에서 기술하는 암세포 또는 암 줄기세포에 의존적인 암의 종류라면 이에 제한되지 아니하며, 바람직하게는 위암일 수 있으나, 이에 제한되는 것은 아니다.
본 발명의 조성물을 이용하는 경우, 위암의 하위 아형으로, 예를 들면, 혼합(mixed stroma) 아형, 위(gastric) 아형, 줄기 유사(stem-like type) 아형, 장(intestinal) 아형 및 염증성(inflammatory) 아형으로 구분할 수 있으며, 특히는 위암의 예후가 가장 좋지 않은 줄기 유사 아형(stem-like type)에 해당하는지 여부를 진단할 수 있다.
본 발명의 조성물에서 상기 단백질의 발현 수준을 측정하는 제제는 상기 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA(peptide nucleic acid) 및 앱타머(aptamer)로 이루어진 군에서 선택된 1종 이상을 포함할 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 "항체"는 항원과 특이적으로 결합하여 항원-항체 반응을 일으키는 물질을 가리킨다. 본 발명의 목적상, 항체는 상기 단백질에 대해 특이적으로 결합하는 항체를 의미한다. 본 발명의 항체는 다클론 항체, 단클론 항체 및 재조합 항체를 모두 포함한다. 상기 항체는 당 업계에 널리 공지된 기술을 이용하여 용이하게 제조될 수 있다. 예를 들어, 다클론 항체는 상기 단백질의 항원을 동물에 주사하고 동물로부터 채혈하여 항체를 포함하는 혈청을 수득하는 과정을 포함하는 당 업계에 널리 공지된 방법에 의해 생산될 수 있다. 이러한 다클론 항체는 염소, 토끼, 양, 원숭이, 말, 돼지, 소, 개 등의 임의의 동물로부터 제조될 수 있다. 또한, 단클론 항체는 당 업계에 널리 공지된 하이브리도마 방법(hybridoma method; Kohler 및 Milstein (1976) European Journal of Immunology 6:511-519 참조), 또는 파지 항체 라이브러리 기술(Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991 참조)을 이용하여 제조될 수 있다. 상기 방법으로 제조된 항체는 겔 전기영동, 투석, 염 침전, 이온교환 크로마토그래피, 친화성 크로마토그래피 등의 방법을 이용하여 분리, 정제될 수 있다. 또한, 본 발명의 항체는 2개의 전장의 경쇄 및 2개의 전장의 중쇄를 갖는 완전한 형태뿐만 아니라, 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란, 적어도 항원 결합 기능을 보유하고 있는 단편을 의미하며, Fab, F(ab'), F(ab')2 및 Fv 등이 있다.
본 발명에서 상기 "올리고펩타이드"는 펩타이드로 2 내지 20 개의 아미노산으로 구성되며 디 펩티드, 트리 펩티드, 테트라 펩티드 및 펜타 펩티드를 포함할 수 있으나, 이에 제한되는 것은 아니다.
본 발명에 상기 "PNA(Peptide Nucleic Acid)"는 인공적으로 합성된, DNA 또는 RNA와 비슷한 중합체를 가리키며, 1991년 덴마크 코펜하겐 대학교의 Nielsen, Egholm, Berg와 Buchardt 교수에 의해 처음으로 소개되었다. DNA는 인산-리보스당 골격을 갖는데 반해, PNA는 펩타이드 결합에 의해 연결된 반복된 N-(2-아미노에틸)-글리신 골격을 가지며, 이로 인해 DNA 또는 RNA에 대한 결합력과 안정성이 크게 증가되어 분자 생물학, 진단 분석 및 안티센스 치료법에 사용되고 있다. PNA는 문헌[Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500]에 상세하게 개시되어 있다.
본 발명에서 상기 "앱타머"는 올리고핵산 또는 펩타이드 분자이며, 앱타머의 일반적인 내용은 문헌[Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24): 142727(1998)]에 상세하게 개시되어 있다.
본 발명의 조성물에서 상기 단백질을 코딩하는 유전자의 발현 수준을 측정하는 제제는 상기 단백질을 코딩하는 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택된 1종 이상을 포함할 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 "프라이머"는 표적 유전자 서열을 인지하는 단편으로서, 정방향 및 역방향의 프라이머 쌍을 포함하나, 바람직하게는, 특이성 및 민감성을 가지는 분석 결과를 제공하는 프라이머 쌍이다. 프라이머의 핵산 서열이 시료 내 존재하는 비-표적 서열과 불일치하는 서열이어서, 상보적인 프라이머 결합 부위를 함유하는 표적 유전자 서열만 증폭하고 비특이적 증폭을 유발하지 않는 프라이머일 때, 높은 특이성이 부여될 수 있다.
본 발명에서 상기 "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 당 업계에서 통상적으로 사용되는 물질로서 제한은 없으나, 바람직하게는 PNA(peptide nucleic acid), LNA(locked nucleic acid), 펩타이드, 폴리펩타이드, 단백질, RNA 또는 DNA일 수 있으며, 가장 바람직하게는 PNA이다. 보다 구체적으로, 상기 프로브는 바이오 물질로서 생물에서 유래되거나 이와 유사한 것 또는 생체 외에서 제조된 것을 포함하는 것으로, 예를 들어, 효소, 단백질, 항체, 미생물, 동식물 세포 및 기관, 신경세포, DNA, 및 RNA일 수 있으며, DNA는 cDNA, 게놈 DNA, 올리고뉴클레오타이드를 포함하며, RNA는 게놈 RNA, mRNA, 올리고뉴클레오타이드를 포함하며, 단백질의 예로는 항체, 항원, 효소, 펩타이드 등을 포함할 수 있다.
본 발명에서 상기 "LNA(Locked nucleic acids)"란, 2'-O, 4'-C 메틸렌 브릿지를 포함하는 핵산 아날로그를 의미한다 [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502]. LNA 뉴클레오사이드는 DNA와 RNA의 일반적 핵산 염기를 포함하며, Watson-Crick 염기 쌍 규칙에 따라 염기 쌍을 형성할 수 있다. 하지만, 메틸렌 브릿지로 인한 분자의 'locking'으로 인해, LNA는 Watson-Crick 결합에서 이상적 형상을 형성하지 못하게 된다. LNA가 DNA 또는 RNA 올리고뉴클레오티드에 포함되면, LNA는 보다 빠르게 상보적 뉴클레오티드 사슬과 쌍을 이루어 이중 나선의 안정성을 높일 수 있다.
본 발명에서 상기 "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.
본 발명에 따른 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택되는 유전자나 이에 의해 코딩되는 단백질의 정보는 알려져 있으므로, 당 업자라면 이를 바탕으로 상기 유전자에 특이적으로 결합하는 프라이머, 프로브 또는 안티센스 뉴클레오티드를 용이하게 디자인할 수 있을 것이다.
본 발명의 다른 구현 예에 따르면, 본 발명의 상기 조성물을 포함하는 암의 예후 예측용 키트에 관한 것이다.
본 발명에서 상기 "키트"는 바이오 마커 성분에 특이적으로 결합하는 프로브 또는 항체를 검출 가능한 표지로 표지하여 바이오 마커의 발현 수준을 평가할 수 있는 도구를 말한다. 프로브 또는 항체 관련하여 검출 가능한 물질을 기질과의 반응에 의해서 직접적으로 표지하는 것뿐만 아니라, 직접적으로 표지된 다른 시약과의 반응성에 의한 발색하는 표지체가 접합된 간접적 표지도 포함한다. 상기 표지체와 발색 반응할 발색 기질 용액, 세척액 및 기타 다른 용액 등을 포함할 수 있으며, 사용되는 시약 성분을 포함하여 제작될 수 있다. 본 발명에서 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있으며, 마커 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브, 반응 완충액, 데옥시뉴클레오티드(dNTPs), Taq-중합효소, 역전사효소, DNase, RNase 억제제, 멸균수 등을 포함할 수 있다. 또한, 키트는 DNA 칩을 수행하기 위해 필요한 필수 요소를 포함하는 암 예후 예측용 유전자를 검출하기 위한 키트일 수 있다. DNA 칩 키트는 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판을 포함하고 기판은 정량 대조군 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있다. 본 발명의 키트는 당 업계에 공지되어 있는 것이라면, 이에 제한되지 않는다.
본 발명에서 상기 키트는 RT-PCR 키트, DNA 칩 키트, ELISA 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(Multiple reaction monitoring) 키트일 수 있다.
본 발명의 상기 키트는 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물, 용액 또는 장치를 더 포함할 수 있다. 예를 들면, 본 발명에서 상기 키트는 역전사 중합효소반응을 수행하기 위해 필요한 필수 요소를 더 포함할 수 있다. 역전사 중합효소반응 키트는 마커 단백질을 코딩하는 유전자에 대해 특이적인 프라이머 쌍을 포함한다. 프라이머는 상기 유전자의 핵산 서열에 특이적인 서열을 가지는 뉴클레오티드로써, 약 7 bp 내지 50 bp의 길이, 보다 바람직하게는 약 10 bp 내지 30 bp의 길이를 가질 수 있다. 또한 대조군 유전자의 핵산 서열에 특이적인 프라이머를 포함할 수 있다. 그 외 역전사 중합효소반응 키트는 테스트 튜브 또는 다른 적절한 용기, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드(dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNase, RNase 억제제 DEPC-수(DEPC-water), 멸균수 등을 포함할 수 있다.
또한, 본 발명의 키트는 DNA 칩을 수행하기 위해 필요한 필수 요소를 포함할 수 있다. DNA 칩 키트는 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드(oligonucleotide)가 부착되어 있는 기판, 및 형광표지 프로브를 제작하기 위한 시약, 제제, 효소 등을 포함할 수 있다. 또한 기판은 대조군 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드를 포함할 수 있다.
또한, 본 발명의 키트는 ELISA를 수행하기 위해 필요한 필수 요소를 포함할 수 있다. ELISA 키트는 상기 단백질에 대해 특이적인 항체를 포함한다. 항체는 마커 단백질에 대한 특이성 및 친화성이 높고 다른 단백질에 대한 교차 반응성이 거의 없는 항체로, 단클론 항체, 다클론 항체 또는 재조합 항체이다. 또한 ELISA 키트는 대조군 단백질에 특이적인 항체를 포함할 수 있다. 그 외 ELISA 키트는 결합된 항체를 검출할 수 있는 시약, 예를 들면, 표지된 2차 항체, 발색단(chromophores), 효소(예: 항체와 컨주게이트됨) 및 그의 기질 또는 항체와 결합할 수 있는 다른 물질 등을 포함할 수 있다.
본 발명의 키트에서 항원-항체 결합반응을 위한 고정체로는 니트로셀룰로오즈 막, PVDF 막, 폴리비닐(polyvinyl) 수지 또는 폴리스티렌(polystyrene) 수지로 합성된 웰 플레이트(Well plate), 유리로 된 슬라이드 글래스 등이 사용될 수 있으나, 이에 제한되는 것은 아니다.
또한, 본 발명의 키트에서 2차 항체의 표지체는 발색 반응을 하는 통상의 발색제가 바람직하며, HRP(horseradish peroxidase), 염기성 탈인산화효소(alkaline phosphatase), 콜로이드 골드(coloid gold), FITC(폴리 L-라이신-플루오르세인 아이소티오시아네이트), RITC(로다민-B-아이소티오시아네이트) 등의 형광물질(fluorescein) 및 색소(dye) 등의 표지체가 사용될 수 있으나, 이에 제한되는 것은 아니다.
또한, 본 발명의 키트에서 발색을 유도하기 위한 발색 기질은 발색 반응을 하는 표지체에 따라 사용하는 것이 바람직하며, TMB(3,3',5,5'-테트라메틸 베지딘), ABTS[2,2'-아지노-비스(3-에틸벤조티아졸린-6-설폰산)], OPD(o-페닐렌다이아민) 등을 사용할 수 있다. 이때, 발색 기질은 완충 용액(0.1 M NaAc, pH 5.5)에 용해된 상태로 제공되는 것이 더욱 바람직하다. TMB와 같은 발색기질은 이차 항체 접합체의 표지체로 사용된 HRP에 의해 분해되어 발색 침적체를 생성하고, 이 발색 침적체의 침적 정도를 육안으로 확인함으로써 상기 마커 단백질들의 존재 유무를 검출한다.
본 발명의 키트에서 세척액은 인산염 완충 용액, NaCl 및 트윈 20(Tween 20)을 포함하는 것이 바람직하며, 0.02 M 인산염 완충용액, 0.13 M NaCl, 및 0.05% 트윈 20으로 구성된 완충 용액(PBST)이 더욱 바람직하다. 세척액은 항원-항체 결합 반응 후 항원-항체 결합체에 2차 항체를 반응시킨 다음 적당량을 고정체에 첨가하여 3 내지 6회 세척한다. 반응 정지 용액은 황산 용액(H2SO4)이 바람직하게 사용될 수 있다.
본 발명의 상기 암, TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코딩되는 단백질 등에 관한 기재는 본 발명의 암의 예후 예측용 조성물에 기재된 바와 중복되어 이하 그 자세한 기재를 생략한다.
본 발명의 또 다른 구현 예에 따르면, 암의 예후 예측을 위한 정보를 제공하는 방법에 관한 것이다.
본 발명의 상기 방법은 상기 목적하는 개체로부터 분리된 생물학적 시료에서 상기 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준을 측정하는 단계를 포함할 수 있다.
본 발명의 상기 방법은 목적하는 개체로부터 분리된 생물학적 시료에서, 암의 예후가 좋지 않거나 좋지 않을 가능성이 높은 지 여부를 선별하기 위한 것일 수 있다.
본 발명에서 상기 목적하는 개체란 암이 발병하였거나 발병 가능성이 높은 개체로, 인간을 포함하는 포유 동물일 수 있고, 예를 들면, 인간, 래트, 마우스, 모르모트, 햄스터, 토끼, 원숭이, 개, 고양이, 소, 말, 돼지, 양 및 염소로 구성된 군으로부터 선택될 수 있고, 바람직하게는 인간일 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 생물학적 시료는 개체로부터 얻어지거나 개체로부터 유래된 임의의 물질, 생물학적 체액, 조직 또는 세포를 의미하는 것으로, 전혈(whole blood), 백혈구(leukocytes), 말초혈액 단핵 세포(peripheral blood mononuclear cells), 백혈구 연층(buffy coat), 혈장(plasma), 혈청(serum), 객담(sputum), 눈물(tears), 점액(mucus), 세비액(nasal washes), 비강 흡인물(nasal aspirate), 호흡(breath), 소변(urine), 정액(semen), 침(saliva), 복강 세척액(peritoneal washings), 복수(ascites), 낭종액(cystic fluid), 뇌척수막 액(meningeal fluid), 양수(amniotic fluid), 선액(glandular fluid), 췌장액(pancreatic fluid), 림프액(lymph fluid), 흉수(pleural fluid), 유두 흡인물(nipple aspirate), 기관지 흡인물(bronchial aspirate), 활액(synovial fluid), 관절 흡인물(joint aspirate), 기관 분비물(organ secretions), 세포(cell), 세포 추출물(cell extract) 및 뇌척수액(cerebrospinal fluid) 등으로 이루어진 군에서 선택된 1종 이상일 수 있으나, 이에 제한되지 않는다.
본 발명에서 상기 단백질의 발현 수준을 측정하는 제제는 상기 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA(peptide nucleic acid) 및 앱타머(aptamer)로 이루어진 군에서 선택된 1종 이상을 포함할 수 있다.
본 발명에서 상기 단백질의 발현 수준의 측정은 단백질 칩 분석, 면역 측정법, 리간드 바인딩 어세이, MALDI-TOF(Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) 분석, SELDI-TOF(Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) 분석, 방사선 면역 분석, 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 블랏팅 또는 ELISA(enzyme linked immunosorbentassay)에 의해 수행될 수 있다.
또한, 본 발명에서 상기 단백질의 발현 수준의 측정은 다중 반응 모니터링 (multiple reaction monitoring; MRM) 방법에 의할 수 있다.
본 발명에서 상기 다중 반응 모니터링 방법 시 내부 표준 물질은 타깃 펩타이드를 구성하는 특정 아미노산을 동위원소로 치환한 합성 펩타이드 또는 대장균 베타 갈락토시다아제를 사용할 수 있다.
본 발명에서 상기 TAGLN 단백질은 서열번호 1로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 CALD1 단백질은 서열번호 2로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 MYL9 단백질은 서열번호 3으로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 MYLK 단백질은 서열번호 4로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 ACTA2 단백질은 서열번호 5로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 TPM2 단백질은 서열번호 6으로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 CNN1 단백질은 서열번호 7로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 MSN 단백질은 서열번호 8로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 CNN3 단백질은 서열번호 9로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 MYH10 단백질은 서열번호 10으로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 MYH11 단백질은 서열번호 11로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 CRIP2 단백질은 서열번호 12로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서 상기 ITGA5 단백질은 서열번호 13으로 표시되는 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.
본 발명에서는 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5의 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준을 측정할 수 있다.
본 발명에서 상기 단백질을 코딩하는 유전자의 발현 수준을 측정하는 제제는 상기 단백질을 코딩하는 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택된 1종 이상을 포함할 수 있다.
본 발명에서 상기 단백질을 코딩하는 유전자의 발현 수준의 측정은 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) 또는 DNA 칩에 의할 수 있다.
본 발명의 정보 제공 방법에서 항체, 올리고펩타이드, 리간드, PNA(peptide nucleic acid), 앱타머(aptamer) 등에 관한 기재와 프라이머, 프로브 등에 관한 기재는 앞서 기재된 바와 중복되어 명세서의 과도한 복잡을 피하기 위하여 이하 그 자세한 기재를 생략한다.
또한, 본 발명에서는 상기 목적하는 개체의 생물학적 시료에 대하여 측정된 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준이 대조군에 비하여 높을 경우, 상기 목적하는 개체에 악성 암으로서 줄기세포의 특성을 갖는 암이 발생하였거나 발생할 가능성이 높은 것으로 예측되어, 암 치료 예후가 좋지 않을 것으로 예측할 수 있다.
본 발명에서 "대조군"이란 암이 발생하지 아니한 정상 대조군이거나, 줄기세포의 특성을 갖지 않는 암세포에서의 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준의 평균 내지 중간 값일 수 있다. 대조군에서의 마커 단백질 또는 이를 코딩하는 핵산분자의 발현량과 분석 대상이 되는 암 환자 유래의 생물학적 시료에서의 마커 단백질 또는 이를 코딩하는 핵산분자의 발현량을 비교할 수 있으며, 상기 발현량의 유의한 변화 여부를 판단하여 악성 암의 발생 또는 발생 가능성 여부를 진단할 수 있다. 상기 정상 대조군 시료의 범위로는 줄기세포의 특성이 없는 것으로 확인된 암 환자 유래의 세포, 이의 배양액, 혈액, 혈청, 혈장, 및 조직도 포함된다.
본 발명에서 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종일 수 있으나, 종양의 분화 및/또는 증식 등 암의 진행이 본 발명에서 기술하는 암 세포 및/또는 암 줄기세포에 의존적인 암의 종류라면 이에 제한되지 않으나, 바람직하게는 위암일 수 있으나 이에 제한되는 것은 아니다.
본 발명에서 상기 방법은 상기에서 측정된 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준으로부터 SMC (smooth mucsle cell) 스코어 값을 계산하는 단계를 추가로 포함할 수 있다. 본 발명의 SMC 스코어 값은 정규화된 유전자 발현 수준을 보기 위해 TopHat-Cufflink14 파이프라인을 사용할 수 있으나 이에 제한되는 것은 아니다. 상기 파이프라인 함수를 이용한 데이터의 처리는 리드 수(Read count) 값을 변위 치환(quantile) 정규화 방법을 통하여 값을 도출할 수 있다. 상기 SMC 스코어 값의 도출은 당업계에서 통상적으로 사용하는 통계 시스템 중에 가장 대표적으로 사용되는 R 패키지 중 EdgeR 패키지를 이용하여 edgeR 사용자의 가이드에 따라 수행될 수 있다(http://www.bioconductor.org/packages/release/bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf). 상기 가이드에 따라 유전자 또는 단백질의 발현 수준을 일반화 선형 모델(Generalized linear models)로부터 로그 강도 값으로 변환시켜 계산할 수 있다.
본 발명에서 상기 SMC 스코어는 상기 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 두 개의 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준, 바람직하게는 로그 강도(log intensity)의 평균 값으로 계산될 수 있고, 보다 바람직하게는 상기 두 개의 유전자의 발현 수준의 로그 강도의 평균 값으로 계산될 수 있다.
다만, 본 발명에서 상기 발현 수준은 하우스키핑(housekeeping) 유전자 또는 이에 의해 코딩되는 단백질에 의해 정규화(normalization)된 값일 수 있으나, 이에 제한되는 것은 아니다. 여기서 상기 하우스키핑 유전자는 β-actin, AHSP, B2M, TUBB2a, GAPDH, HBS1L, HPRT1 또는 SDHA 등이 있을 수 있으나 이에 제한되는 것은 아니다.
본 발명의 일 예시에서, 암의 예후를 예측하는 방법은 계산된 SMC 스코어 값이 5 내지 15의 값 이상인 경우, 바람직하게는 10 내지 12의 값 이상인 경우, 보다 바람직하게는 11 이상인 경우, 암 줄기세포를 포함하는 악성 암의 발병 또는 발병 가능성이 높아 암 환자의 치료 예후가 좋지 않거나, 암 환자의 생존율이 낮거나 생존 기간이 짧을 것으로 예측하는 단계를 추가로 포함할 수 있다.
본 발명의 다른 예시에서, 본 발명의 암의 예후를 예측하는 방법은 계산된 SMC 스코어 값이 5 내지 15의 값 미만인 경우, 바람직하게는 10 내지 12의 값 미만인 경우, 보다 바람직하게는 11 미만인 경우, 암 환자의 치료 예후가 좋거나, 암 환자의 생존율이 높거나 생존 기간이 길 것으로 예측하는 단계를 추가로 포함할 수 있다.
본 발명의 상기 암, TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코딩되는 단백질 등에 관한 기재는 본 발명의 암 진단용 조성물에 기재된 바와 중복되어 이하 그 자세한 기재를 생략한다.
본 발명의 또 다른 구현 예에 따르면, 암의 예후 진단기기에 관한 것이다.
본 발명의 상기 진단기기는 목적하는 개체로부터 얻어진 생물학적 시료로부터 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준을 측정하는 측정부; 및 상기 측정부에서 측정된 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준으로부터 SMC 스코어 값을 계산하는 연산부를 포함할 수 있다.
본 발명의 상기 진단기기의 연산부는 상기 측정부에서 측정된 유전자 또는 단백질의 발현 수준의 로그 강도 값의 평균 (average of log intensity)으로 SMC 스코어 값을 계산하는 기능을 수행하는 것일 수 있다.
본 발명의 상기 진단기기의 연산부에서 도출된 SMC 스코어 값으로부터 목적하는 개체의 암의 치료 예후를 예측할 수 있다. 보다 상세하게는 상기 연산부에서는 도출된 SMC 스코어의 값에 따라 암의 예후 예측에 관한 정보를 생성하여 분류함으로써 악성 암으로서 줄기세포의 특성을 가진 암으로 좋지 않은 예후가 예견되는 목적하는 개체를 결정할 수 있다.
본 발명의 일 예시에서, 상기 연산부에서는 계산된 SMC 스코어 값이 5 내지 15의 값 이상인 경우, 바람직하게는 10 내지 12의 값 이상인 경우, 보다 바람직하게는 11 이상인 경우 암 줄기세포를 포함하는 악성 암의 발병 또는 발병 가능성이 높아 암 환자의 치료 예후가 좋지 않을 것으로 예측하여 출력하는 출력부를 더 포함할 수 있다.
본 발명의 다른 예시에서, 상기 연산부에서는 계산된 SMC 스코어 값이 5 내지 15의 값 미만인 경우, 바람직하게는 10 내지 12의 값 미만인 경우, 보다 바람직하게는 11 미만인 경우 암 환자의 치료 예후가 좋을 것으로 예측하여 출력할 수 있다.
본 발명의 상기 진단기기는 연산부에서 예측된 목적하는 개체의 암의 예후를 출력하는 출력부를 추가로 포함할 수 있다.
본 발명의 상기 암, TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코딩되는 단백질 등에 관한 기재는 본 발명의 암 진단용 조성물에 기재된 바와 중복되어 이하 그 자세한 기재를 생략한다.
본 발명에 따른 조성물 및 방법을 이용하는 경우 악성 암, 특히는 악성 위암을 진단함으로써, 위암의 예후를 예측할 수 있다.
도 1은 본 발명의 일 실시예에 따른 연세 코호트에서 위암 환자 (n = 497)의 종양에 대한 전사체 (transcriptome) 데이터를 분석한 결과를 나타낸 도이다.
도 2는 본 발명의 일 실시예에 따른 줄기 유사 GC 아형과 장 GC 아형 환자의 생존율과 유전자세트 증폭분석 (Gene Set Enrichment Analysis; GSEA) 결과를 나타낸 도이다.
도 3a는 본 발명의 일 실시예에 따른 SFRP4 마이크로어레이 데이터를 로그값으로 변환한 결과를 나타낸 도이다.
도 3b는 본 발명의 일 실시예에 따른 낮은 SMC (SMC-low) 환자 군과 높은 SMC (SMC-high) 환자 군에서 EMT 시그니처 유전자의 히트맵 결과를 나타낸 도이다.
도 4a는 본 발명의 일 실시예에 따른 위암에서 SFRP4와 SMC 스코어를 구성하는 SMC 유전자들의 공동 발현 (Co-expression) 스코어를 확인한 결과를 나타낸 도이다.
도 4b는 본 발명의 일 실시예에 따른 위암 데이터 세트 (The Cancer Genome Atlas Stomach adenocarcinoma; TCGA STAD)에서 SFRP4와 SMC 유전자 결과를 나타낸 도이다.
도 5는 본 발명의 일 실시예에 따른 높은 SFRP4 (SFRP4-high) 군, 낮은 SFRP-4 (SFRP4-low) 군, 낮은 SMC (SMC-low) 환자 군과 높은 SMC (SMC-high) 환자 군의 카플란-마이어 생존 분석 결과를 나타낸 도이다.
도 6은 본 발명의 일 실시예에 따른 암의 병기에 따른 SMC 스코어 값을 비교한 결과를 나타낸 도이다.
도 7은 본 발명의 일 실시예에 따른 주성분 분석 (Principal Component Analysis; PCA)을 통해 DEG 유전자의 발현 프로파일을 기반으로 GC 세포주인 클러스터 A (HS746T 및 MKN1 세포주)와 클러스터 B (SNU601 및 NCIN87 세포주)를 나누어 산점도를 확인한 결과를 나타낸 도이다.
도 8a는 본 발명의 일 실시예에 따른 위암 (GC tumor 및 GC cell line)의 유전자세트 증폭분석 (GSEA) 결과와 각 클러스터의 SMC 스코어 값을 비교한 결과를 나타낸 도이다.
도 8b는 본 발명의 일 실시예에 따른 다양한 위암 세포주들에서 분석된 RNA-시퀀싱 데이터를 히트맵으로 나타낸 도이다.
도 9는 본 발명의 일 실시예에 따라 선별된 SMC 스코어를 도출하기 위한 13 개 유전자 각각을 통해 줄기 유사 아형을 구분할 수 있음을 확인한 결과를 나타낸 도이다.
도 10은 본 발명의 일 실시예에 따른 TCGA STAD에서 낮은 SMC (SMC-low) 환자 군과 높은 SMC (SMC-high) 환자 군에서 SRF 모티프 타겟 유전자의 히트맵 결과를 나타낸 도이다.
도 11a는 본 발명의 일 실시예에 따른 TAGLN 프로모터 형질 감염시킨 HS746T 및 MKN1 세포주에서 루시퍼레이즈 어세이(Luciferase reporter assay)를 통하여 확인한 결과를 나타낸 도이다.
도 11b는 본 발명의 일 실시예에 따른 장 세포와 유사한 비 EMT 세포주와 줄기 세포와 유사한 EMT 세포 라인에서 MYOCD와 MRTFA, MRTFB(myocardin-related factor A and B)의 발현 레벨을 확인한 결과를 나타낸 도이다.
도 11c는 본 발명의 일 실시예에 따른 HS746T 및 MKN1 세포주에 MRTFA를 넉다운 시킨 후 SMC 유전자의 발현 정도를 RT-PCR로 확인한 결과를 나타낸 도이다.
도 12는 본 발명의 일 실시예에 따른 세포 라인 별로 13 개의 SMC 스코어 유전자의 mRNA 발현 수준을 로그 강도 값 (log2 intensity value)으로 계산하여 비교 확인한 결과를 나타낸 도이다.
도 2는 본 발명의 일 실시예에 따른 줄기 유사 GC 아형과 장 GC 아형 환자의 생존율과 유전자세트 증폭분석 (Gene Set Enrichment Analysis; GSEA) 결과를 나타낸 도이다.
도 3a는 본 발명의 일 실시예에 따른 SFRP4 마이크로어레이 데이터를 로그값으로 변환한 결과를 나타낸 도이다.
도 3b는 본 발명의 일 실시예에 따른 낮은 SMC (SMC-low) 환자 군과 높은 SMC (SMC-high) 환자 군에서 EMT 시그니처 유전자의 히트맵 결과를 나타낸 도이다.
도 4a는 본 발명의 일 실시예에 따른 위암에서 SFRP4와 SMC 스코어를 구성하는 SMC 유전자들의 공동 발현 (Co-expression) 스코어를 확인한 결과를 나타낸 도이다.
도 4b는 본 발명의 일 실시예에 따른 위암 데이터 세트 (The Cancer Genome Atlas Stomach adenocarcinoma; TCGA STAD)에서 SFRP4와 SMC 유전자 결과를 나타낸 도이다.
도 5는 본 발명의 일 실시예에 따른 높은 SFRP4 (SFRP4-high) 군, 낮은 SFRP-4 (SFRP4-low) 군, 낮은 SMC (SMC-low) 환자 군과 높은 SMC (SMC-high) 환자 군의 카플란-마이어 생존 분석 결과를 나타낸 도이다.
도 6은 본 발명의 일 실시예에 따른 암의 병기에 따른 SMC 스코어 값을 비교한 결과를 나타낸 도이다.
도 7은 본 발명의 일 실시예에 따른 주성분 분석 (Principal Component Analysis; PCA)을 통해 DEG 유전자의 발현 프로파일을 기반으로 GC 세포주인 클러스터 A (HS746T 및 MKN1 세포주)와 클러스터 B (SNU601 및 NCIN87 세포주)를 나누어 산점도를 확인한 결과를 나타낸 도이다.
도 8a는 본 발명의 일 실시예에 따른 위암 (GC tumor 및 GC cell line)의 유전자세트 증폭분석 (GSEA) 결과와 각 클러스터의 SMC 스코어 값을 비교한 결과를 나타낸 도이다.
도 8b는 본 발명의 일 실시예에 따른 다양한 위암 세포주들에서 분석된 RNA-시퀀싱 데이터를 히트맵으로 나타낸 도이다.
도 9는 본 발명의 일 실시예에 따라 선별된 SMC 스코어를 도출하기 위한 13 개 유전자 각각을 통해 줄기 유사 아형을 구분할 수 있음을 확인한 결과를 나타낸 도이다.
도 10은 본 발명의 일 실시예에 따른 TCGA STAD에서 낮은 SMC (SMC-low) 환자 군과 높은 SMC (SMC-high) 환자 군에서 SRF 모티프 타겟 유전자의 히트맵 결과를 나타낸 도이다.
도 11a는 본 발명의 일 실시예에 따른 TAGLN 프로모터 형질 감염시킨 HS746T 및 MKN1 세포주에서 루시퍼레이즈 어세이(Luciferase reporter assay)를 통하여 확인한 결과를 나타낸 도이다.
도 11b는 본 발명의 일 실시예에 따른 장 세포와 유사한 비 EMT 세포주와 줄기 세포와 유사한 EMT 세포 라인에서 MYOCD와 MRTFA, MRTFB(myocardin-related factor A and B)의 발현 레벨을 확인한 결과를 나타낸 도이다.
도 11c는 본 발명의 일 실시예에 따른 HS746T 및 MKN1 세포주에 MRTFA를 넉다운 시킨 후 SMC 유전자의 발현 정도를 RT-PCR로 확인한 결과를 나타낸 도이다.
도 12는 본 발명의 일 실시예에 따른 세포 라인 별로 13 개의 SMC 스코어 유전자의 mRNA 발현 수준을 로그 강도 값 (log2 intensity value)으로 계산하여 비교 확인한 결과를 나타낸 도이다.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
준비예 1: 위암 환자의 데이터 모집
본 발명자들은 연세 암 센터 (대한민국 서울)에서 치료 목적의 위 절제술 (curative intent gastrectomy)을 받은 위암 환자로부터 신선한 냉동 종양 조직을 확보하고 임상 데이터를 일치시켰다. 본 연구는 연세대학교 의과대학 평가위원회 (Institutional Review Board; IRB)의 승인을 얻어 모든 실험을 수행하였으며, 모든 샘플은 환자로부터 서면 동의를 얻은 후 수집되었다.
준비예 2: 세포의 배양
본 발명의 일 실시예에 따른 MKN1, SNU601, NCIN87 세포주는 세포주 은행의 가이드에 따라 10 % 소 태아 혈청 (FBS), 2mM L- 글루타민, 100 U/ml 페니실린 및 100 μg/ml 스트렙토 마이신이 함유된 RPMI1640에서 배양되었으며, HS746T 세포주도 세포주 은행의 가이드에 따라 10 % FBS, 2mM L- 글루타민, 100 U/ml 페니실린 및 100 μg/ml 스트렙토 마이신을 포함하는 DMEM에서 배양되었고, 모든 세포주는 37 ℃, 5 % CO2 인큐베이터에서 배양하여 오염도 테스트 후 실험에 이용하였다.
실시예 1: SMC 유전자의 선별
위암 (gastric cancer; GC) 환자를 임상적으로 검증된 분류 체계에 따라 5 가지 하위 아형 (혼합, 위, 줄기 유사, 장 및 염증)으로 나누었다. 줄기 유사 아형에서 최악의 예후를 보였고, 줄기 유사 암 세포의 분자적 특성을 알아보기 위하여 상기 준비예 1에서 수득한 연세 코호트 위암 환자 (n = 497) 샘플의 전사체를 분석하였다. 혼합 (n = 99), 위 (n = 89), 줄기 유사 (n = 117), 장 (n = 102) 및 염증 (n = 90)의 하위 유형에 따라 SMC 유전자의 발현 수준을 비교한 결과를 도 1에 나타내었으며, 줄기 유사 GC 아형 (n = 117)을 가진 환자와 장 GC 아형 (n = 102)을 가진 환자의 생존율 및 유전자세트 증폭분석 (Gene Set Enrichment Analysis; GSEA) 결과를 비교하여 도 2에 나타내었다.
그 결과, 흥미롭게도 생존율이 낮은 줄기 유사 GC 아형 (n = 117)을 가진 환자는 실질적으로 다른 유전적 및 분자적 특징을 보인 장 GC 아형 (n = 102)을 가진 환자에 비해 평활근 특이적 유전자(smooth muscle-specific genes)의 높은 발현을 나타내는 것을 확인할 수 있었다. 도 2에서처럼 생존율이 좋지 않은 줄기 유사 GC 아형 환자군에서 SMC 유전자들이 상향 조절(up-regulation)되어 있음을 알 수 있다(도 1 참조).
줄기 유사 그룹에서 높게 나타나는 임상 마커인 SFRP4의 발현 수준에 의해 서브타이핑 방법(subtyping method)을 통해 검증하였고, 낮은 SMC (SMC-low) 환자 군과 높은 SMC (SMC-high) 환자 군에서 EMT 시그니처 유전자의 히트맵 결과를 확인하였다(도 3a 및 도 3b 참조). 상기 SFRP4(secreted frizzled-related protein 4)는 악성 암이거나 표준 화학 치료에 반응하지 않는 좋지 않은 예후와 관련이 있음을 나타내는 임상 마커에 해당한다. 본 발명자들은 SMC(smooth muscle cell) 유전자를 선별하기 위하여 SFRP4와 SMC 유전자들의 공동 발현 (Co-expression) 스코어를 확인하여 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5를 선별할 수 있었다(도 4a 참조). 상기와 같은 공동 발현 데이터베이스로부터 SFRP4 발현과 37 개의 인간 위암 (GC) 데이터 세트(human GC public datasets)에서의 SMC 마커와 상관 관계가 있음을 확인하였다. 도 4b를 참조하면 암 유전체 아틀라스 (The Cancer Genome Atlas; TCGA)의 위 종양 데이터 세트에서 SFRP4 전사체를 분석한 결과 상관 관계를 확인할 수 있었다.
도 5를 참조하면, 높은 SFRP4 (SFRP4-high) 군, 낮은 SFRP-4 (SFRP4-low) 군, 낮은 SMC (SMC-low) 환자 군과 높은 SMC (SMC-high) 환자 군의 카플란-마이어 생존 분석한 결과, TCGA 위암 데이터베이스에서 SMC 점수는 높은 SFRP4 군보다 예후가 매우 좋지 않은 환자 군을 더 명확하게 구분하는 것으로 확인되어 SMC 유전자의 발현이 암의 악성 예후와 관련이 있음을 시사한다(도 5 참조).
근육층 및 주변 연조직으로의 종양 침습이 SMC 점수에 영향을 미칠 가능성을 배제하기 위해 TCGA에서 위암 환자를 종양 병기 및 조직 학적 유형에 따라 분류하였으며, 진단 당시의 단계와 SMC 점수 사이에는 상관 관계가 없는 것을 확인하였다. 그와 달리 SMC 점수는 조직학적 유형에 따라 다르며 확산 조직학에서 높았다(도 6 참조). 따라서 줄기와 유사한 GC는 SMC 유전자의 상향 조절과 함께 단계 독립적인 방식으로 불량한 예후를 보였다.
실시예 2: 선별된 SMC 유전자의 발현과 악성 형질과의 관계 확인
종양 조직의 대량 분석 (bulk analysis)은 비암성 세포 (non-cancerous cells), 특히 간질 근육 세포 (stromal muscle cells)에 의한 불필요한 오염을 유발하여 전사체 분석 결과에 영향을 미칠 수 있어 SMC 유전자의 발현이 간질 근육 세포 때문이 아니라 암 세포에 내재적임을 확인하는 것이 중요하다. 이를 위해 위암 세포주에 대한 RNA-seq 분석을 수행하고 전사적 특성에 따라 세포주를 그룹화하였으며 주성분 분석 (Principal Component Analysis; PCA)을 통해 줄기 유사 환자에서 상향 조절된 차등적으로 발현된 유전자 (DEG)의 발현 프로파일을 기반으로 GC 세포주의 산점도를 확인하였다(도 7 참조). 클러스터 A의 세포주는 유전자 세트 농축 분석에서 GC 환자에서와 같이 클러스터 B의 세포주에 비해 평활근 수축 경로에서 현저한 상승이 나타나는 것을 확인할 수 있었다(도 8a 및 도 8b 참조). 참고로, 클러스터 A 세포주는 EMT-서브타입 GC (EMT-subtype GC) 라인이고, 클러스터 B는 장 GC(intestinal GC) 유사한 비 EMT 세포주 (non-EMT cell lines)에 해당한다. 상기와 같은 결과로부터 세포주와 환자 데이터 사이에는 유사한 경향을 띠는 결과가 나타나는 것을 확인할 수 있으며, 클러스터 A와 클러스터 B 세포주 간에는 차별화되어 나타나는 것을 확인할 수 있다. 즉, 줄기 유사 세포인 클러스터 A에서 SMC 스코어 값이 높게 나타나며, 선별된 SMC 유전자가 높게 발현되므로 상기 유전자와 악성 위암과의 상관 관계를 알 수 있다. 이와 함께 선별된 SMC 유전자 각각으로부터 위암의 예후가 가장 좋지 않은 줄기 유사 아형(stem like type)을 구분할 수 있음을 추가로 검증하였다(도 9 참조).
실시예 3: MRTFA-SRF 전사 재프로그래밍(MRTFA-SRF transcriptional reprogramming)과 SMC 유전자와의 관계
연세 코호트에서 수집된 GC 환자의 전사 프로필을 확인하였으며, 평활근에서 SMC 유전자는 전사 메커니즘에서 미오카르딘 (myocardin)의 의존성 여부로 2 부류로 나누었다. 계층적 클러스터링을 기반으로 한 줄기 유사 GC에서 미오카르딘 의존성 SMC 유전자 (myocardin-dependent SMC gene)의 상향 조절이 미오카르딘 비의존성 SMC 유전자 (myocardin-independent SMC gene)의 상향 조절보다 더 두드러지게 나타났다. 미오카르딘-의존성 유전자는 줄기 유사 아형 환자 mRNA 발현 수준이 증가됨과 함께 성공적으로 그룹화된 반면, 미오카르딘-비의존성 유전자는 줄기 유사 아형 환자를 그룹화되지 않았고 mRNA 발현 수준의 상향 조절도 나타나지 않았다.
미오카르딘-SRF 전사 인자 복합체의 중요성에 대한 더 자세한 정보를 위해, HS746T, MKN1, KATOIII, NCIN87 세포주의 전사체로 DNA 모티프 활성 분석을 수행하였으며, 이를 예측하는 ISMARA (Integrated System for Motif Activity Response Analysis) 알고리즘을 사용하여 조절 모티프 및 관련 표적 유전자를 예측하였다. ISMARA에 따르면, SRF10의 DNA 모티프인 CArG 박스를 통해 106 개의 유전자가 유도되었으며, 이 유전자의 대부분은 액틴 관련 및 근육 구조 발달 경로에 관여한다. SRF 모티프 표적 유전자는 또한 ACTG1, CNN1, CNN2, MYH3, MYH9, MYL9, MYLK, TAGLN, TPM2 및 VCL과 같은 여러 SMC 유전자 마커를 포함하고 있으며, SRF 모티프 표적 유전자의 발현은 줄기 유사 GC 세포주에서 상향 조절되는 것으로 확인되었다. GC 환자 중 연세 코호트의 줄기 유사 환자와 TCGA의 높은 SMC 점수 환자는 SRF 모티프 유전자에 따라 클러스터링되었다(도 10 참조).
다음으로, 본 발명자는 transgelin (TAGLN) 프로모터에서 두 개의 CArG 박스 (CArG box)를 돌연변이 시켰다. 참고로, SMC 스코어를 위한 유전자 성분인 TAGLN은 혈관 평활근 세포에서 발현되는 액틴 결합 단백질로 TGFβ- 유도성으로도 알려져 있다. 루시퍼레이스 활성 분석 (luciferase activity assay) 결과 CArG 박스 제거 (CArG box ablation) 시 TAGLN 프로모터 활성을 유의하게 억제하였다. 그러나 SMAD 결합 요소 (SMAD binding element; SBE)의 추가 절제는 그 정도까지 활성에 영향을 미치지 않는 것으로 나타났으며, 이는 SBE가 아닌 CArG 박스가 줄기 유사 아형 GC 세포주에서 SMC 유전자의 유도에 중요함을 시사한다(도 11a 참조).
그러나, 우리는 GC 세포주가 평활근 및 심장 근육 세포 (smooth and cardiac muscle cell)에서 SMC 유전자 발현의 마스터 조절자 (master regulator)인 미오카르딘을 발현하지 않는다는 것을 발견했으며 그 대신 미오카르딘 계열의 전사 활성자(coactivators)로서 미오카르딘 관련 전사 인자-A (myocardin-related transcription factor-A; MRTFA) 및 미오카르딘 관련 전사 인자-B (myocardin-related transcription factor-B; MRTFB)가 GC 세포주에 존재하면서 미오카르딘과 상동성을 가진다(도 11b 참조). 특히, MRTFA의 발현 수준은 장 세포주에서보다 줄기 유사에서 더 높았으며, 이는 MRTFA가 GC 세포에서 SMC 유전자의 전사 조절자일 가능성을 시사한다(도 11b 참조). 더욱이, MRTFA 녹다운은 SMC 유전자 발현 수준을 상당히 감소시키는 것으로 확인되었다(도 11c 참조).
실시예 4: SMC 스코어의 도출 및 검증
위암 줄기세포 특성을 갖는 클러스터 A군(HS746T 및 MKN1 세포주)과 일반 암세포의 특성을 갖는 클러스터 B군(YCC7 및 NCIN87)에서 측정된 SMC 스코어 값을 비교하여, SMC 스코어 값에 따른 불량한 예후를 가진 위암을 진단 또는 예측할 수 있음을 확인하고, 환자 샘플군을 통해 검증하고자 하였다.
상기 준비예에서 수득한 위 절제술을 받은 위암 환자로부터 신선한 냉동 종양 조직을 확보하여 임상 데이터를 일치시켰다. 그 후 샘플을 사용하여 두 군 (batches)의 코호트 데이터 세트 (n = 497, GSE13861 및 GSE84437)를 생성하였고, Illumina HUmanHT-12 v3.0 Expression BeacChip 어레이를 통해 마이크로 어레이 분석을 수행하였다. mRNA 발현 수준은 로그 강도 값 (log intensity value)으로 표시되었으며 SMC 스코어는 13 개의 SMC 스코어 유전자 (TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2, ITGA5)의 로그 강도의 평균으로 계산되었다. SMC 스코어가 높은 환자의 컷오프 값은 11임을 최종적으로 도출하였다(도 12 참조). 상기 결과를 통해 본 발명에 따른 SMC 스코어 값이 11 이상에 해당할 경우 위암의 예후가 매우 좋지 않을 것임을 효과적으로 예측할 수 있다.
상기 결과를 종합하면, 본 발명자들은 줄기 유사 아형의 GC가 수축성 평활근과 유사한 분자 및 대사 특성을 나타내며, 높은 SMC 스코어 값을 가지며, SMC 스코어 값으로 특히는 11 이상에 해당할 경우 위암의 예후가 매우 좋지 않은 상관 관계가 있음을 확인하였다. SMC 스코어 값의 계산을 통하여 약물 내성, 전이, 대사 스트레스에서의 생존 및 증식 감소와 관련된 SMC 특성을 획득하였는지 여부로 악성 위암의 예후를 진단할 수 있다.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
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Glu Ala Glu Lys Leu Ala Lys Glu Arg Gln Glu Ala Glu Glu Ala Lys
385 390 395 400
Glu Ala Leu Leu Gln Ala Ser Arg Asp Gln Lys Lys Thr Gln Glu Gln
405 410 415
Leu Ala Leu Glu Met Ala Glu Leu Thr Ala Arg Ile Ser Gln Leu Glu
420 425 430
Met Ala Arg Gln Lys Lys Glu Ser Glu Ala Val Glu Trp Gln Gln Lys
435 440 445
Ala Gln Met Val Gln Glu Asp Leu Glu Lys Thr Arg Ala Glu Leu Lys
450 455 460
Thr Ala Met Ser Thr Pro His Val Ala Glu Pro Ala Glu Asn Glu Gln
465 470 475 480
Asp Glu Gln Asp Glu Asn Gly Ala Glu Ala Ser Ala Asp Leu Arg Ala
485 490 495
Asp Ala Met Ala Lys Asp Arg Ser Glu Glu Glu Arg Thr Thr Glu Ala
500 505 510
Glu Lys Asn Glu Arg Val Gln Lys His Leu Lys Ala Leu Thr Ser Glu
515 520 525
Leu Ala Asn Ala Arg Asp Glu Ser Lys Lys Thr Ala Asn Asp Met Ile
530 535 540
His Ala Glu Asn Met Arg Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg
545 550 555 560
Gln Ile Arg Gln Gly Asn Thr Lys Gln Arg Ile Asp Glu Phe Glu Ser
565 570 575
Met
<210> 9
<211> 329
<212> PRT
<213> Homo sapiens
<400> 9
Met Thr His Phe Asn Lys Gly Pro Ser Tyr Gly Leu Ser Ala Glu Val
1 5 10 15
Lys Asn Lys Ile Ala Ser Lys Tyr Asp His Gln Ala Glu Glu Asp Leu
20 25 30
Arg Asn Trp Ile Glu Glu Val Thr Gly Met Ser Ile Gly Pro Asn Phe
35 40 45
Gln Leu Gly Leu Lys Asp Gly Ile Ile Leu Cys Glu Leu Ile Asn Lys
50 55 60
Leu Gln Pro Gly Ser Val Lys Lys Val Asn Glu Ser Ser Leu Asn Trp
65 70 75 80
Pro Gln Leu Glu Asn Ile Gly Asn Phe Ile Lys Ala Ile Gln Ala Tyr
85 90 95
Gly Met Lys Pro His Asp Ile Phe Glu Ala Asn Asp Leu Phe Glu Asn
100 105 110
Gly Asn Met Thr Gln Val Gln Thr Thr Leu Val Ala Leu Ala Gly Leu
115 120 125
Ala Lys Thr Lys Gly Phe His Thr Thr Ile Asp Ile Gly Val Lys Tyr
130 135 140
Ala Glu Lys Gln Thr Arg Arg Phe Asp Glu Gly Lys Leu Lys Ala Gly
145 150 155 160
Gln Ser Val Ile Gly Leu Gln Met Gly Thr Asn Lys Cys Ala Ser Gln
165 170 175
Ala Gly Met Thr Ala Tyr Gly Thr Arg Arg His Leu Tyr Asp Pro Lys
180 185 190
Met Gln Thr Asp Lys Pro Phe Asp Gln Thr Thr Ile Ser Leu Gln Met
195 200 205
Gly Thr Asn Lys Gly Ala Ser Gln Ala Gly Met Leu Ala Pro Gly Thr
210 215 220
Arg Arg Asp Ile Tyr Asp Gln Lys Leu Thr Leu Gln Pro Val Asp Asn
225 230 235 240
Ser Thr Ile Ser Leu Gln Met Gly Thr Asn Lys Val Ala Ser Gln Lys
245 250 255
Gly Met Ser Val Tyr Gly Leu Gly Arg Gln Val Tyr Asp Pro Lys Tyr
260 265 270
Cys Ala Ala Pro Thr Glu Pro Val Ile His Asn Gly Ser Gln Gly Thr
275 280 285
Gly Thr Asn Gly Ser Glu Ile Ser Asp Ser Asp Tyr Gln Ala Glu Tyr
290 295 300
Pro Asp Glu Tyr His Gly Glu Tyr Gln Asp Asp Tyr Pro Arg Asp Tyr
305 310 315 320
Gln Tyr Ser Asp Gln Gly Ile Asp Tyr
325
<210> 10
<211> 1976
<212> PRT
<213> Homo sapiens
<400> 10
Met Ala Gln Arg Thr Gly Leu Glu Asp Pro Glu Arg Tyr Leu Phe Val
1 5 10 15
Asp Arg Ala Val Ile Tyr Asn Pro Ala Thr Gln Ala Asp Trp Thr Ala
20 25 30
Lys Lys Leu Val Trp Ile Pro Ser Glu Arg His Gly Phe Glu Ala Ala
35 40 45
Ser Ile Lys Glu Glu Arg Gly Asp Glu Val Met Val Glu Leu Ala Glu
50 55 60
Asn Gly Lys Lys Ala Met Val Asn Lys Asp Asp Ile Gln Lys Met Asn
65 70 75 80
Pro Pro Lys Phe Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu
85 90 95
Asn Glu Ala Ser Val Leu His Asn Leu Lys Asp Arg Tyr Tyr Ser Gly
100 105 110
Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val Ile Asn Pro Tyr
115 120 125
Lys Asn Leu Pro Ile Tyr Ser Glu Asn Ile Ile Glu Met Tyr Arg Gly
130 135 140
Lys Lys Arg His Glu Met Pro Pro His Ile Tyr Ala Ile Ser Glu Ser
145 150 155 160
Ala Tyr Arg Cys Met Leu Gln Asp Arg Glu Asp Gln Ser Ile Leu Cys
165 170 175
Thr Gly Glu Ser Gly Ala Gly Lys Thr Glu Asn Thr Lys Lys Val Ile
180 185 190
Gln Tyr Leu Ala His Val Ala Ser Ser His Lys Gly Arg Lys Asp His
195 200 205
Asn Ile Pro Gly Glu Leu Glu Arg Gln Leu Leu Gln Ala Asn Pro Ile
210 215 220
Leu Glu Ser Phe Gly Asn Ala Lys Thr Val Lys Asn Asp Asn Ser Ser
225 230 235 240
Arg Phe Gly Lys Phe Ile Arg Ile Asn Phe Asp Val Thr Gly Tyr Ile
245 250 255
Val Gly Ala Asn Ile Glu Thr Tyr Leu Leu Glu Lys Ser Arg Ala Val
260 265 270
Arg Gln Ala Lys Asp Glu Arg Thr Phe His Ile Phe Tyr Gln Leu Leu
275 280 285
Ser Gly Ala Gly Glu His Leu Lys Ser Asp Leu Leu Leu Glu Gly Phe
290 295 300
Asn Asn Tyr Arg Phe Leu Ser Asn Gly Tyr Ile Pro Ile Pro Gly Gln
305 310 315 320
Gln Asp Lys Asp Asn Phe Gln Glu Thr Met Glu Ala Met His Ile Met
325 330 335
Gly Phe Ser His Glu Glu Ile Leu Ser Met Leu Lys Val Val Ser Ser
340 345 350
Val Leu Gln Phe Gly Asn Ile Ser Phe Lys Lys Glu Arg Asn Thr Asp
355 360 365
Gln Ala Ser Met Pro Glu Asn Thr Val Ala Gln Lys Leu Cys His Leu
370 375 380
Leu Gly Met Asn Val Met Glu Phe Thr Arg Ala Ile Leu Thr Pro Arg
385 390 395 400
Ile Lys Val Gly Arg Asp Tyr Val Gln Lys Ala Gln Thr Lys Glu Gln
405 410 415
Ala Asp Phe Ala Val Glu Ala Leu Ala Lys Ala Thr Tyr Glu Arg Leu
420 425 430
Phe Arg Trp Leu Val His Arg Ile Asn Lys Ala Leu Asp Arg Thr Lys
435 440 445
Arg Gln Gly Ala Ser Phe Ile Gly Ile Leu Asp Ile Ala Gly Phe Glu
450 455 460
Ile Phe Glu Leu Asn Ser Phe Glu Gln Leu Cys Ile Asn Tyr Thr Asn
465 470 475 480
Glu Lys Leu Gln Gln Leu Phe Asn His Thr Met Phe Ile Leu Glu Gln
485 490 495
Glu Glu Tyr Gln Arg Glu Gly Ile Glu Trp Asn Phe Ile Asp Phe Gly
500 505 510
Leu Asp Leu Gln Pro Cys Ile Asp Leu Ile Glu Arg Pro Ala Asn Pro
515 520 525
Pro Gly Val Leu Ala Leu Leu Asp Glu Glu Cys Trp Phe Pro Lys Ala
530 535 540
Thr Asp Lys Thr Phe Val Glu Lys Leu Val Gln Glu Gln Gly Ser His
545 550 555 560
Ser Lys Phe Gln Lys Pro Arg Gln Leu Lys Asp Lys Ala Asp Phe Cys
565 570 575
Ile Ile His Tyr Ala Gly Lys Val Asp Tyr Lys Ala Asp Glu Trp Leu
580 585 590
Met Lys Asn Met Asp Pro Leu Asn Asp Asn Val Ala Thr Leu Leu His
595 600 605
Gln Ser Ser Asp Arg Phe Val Ala Glu Leu Trp Lys Asp Val Asp Arg
610 615 620
Ile Val Gly Leu Asp Gln Val Thr Gly Met Thr Glu Thr Ala Phe Gly
625 630 635 640
Ser Ala Tyr Lys Thr Lys Lys Gly Met Phe Arg Thr Val Gly Gln Leu
645 650 655
Tyr Lys Glu Ser Leu Thr Lys Leu Met Ala Thr Leu Arg Asn Thr Asn
660 665 670
Pro Asn Phe Val Arg Cys Ile Ile Pro Asn His Glu Lys Arg Ala Gly
675 680 685
Lys Leu Asp Pro His Leu Val Leu Asp Gln Leu Arg Cys Asn Gly Val
690 695 700
Leu Glu Gly Ile Arg Ile Cys Arg Gln Gly Phe Pro Asn Arg Ile Val
705 710 715 720
Phe Gln Glu Phe Arg Gln Arg Tyr Glu Ile Leu Thr Pro Asn Ala Ile
725 730 735
Pro Lys Gly Phe Met Asp Gly Lys Gln Ala Cys Glu Arg Met Ile Arg
740 745 750
Ala Leu Glu Leu Asp Pro Asn Leu Tyr Arg Ile Gly Gln Ser Lys Ile
755 760 765
Phe Phe Arg Ala Gly Val Leu Ala His Leu Glu Glu Glu Arg Asp Leu
770 775 780
Lys Ile Thr Asp Ile Ile Ile Phe Phe Gln Ala Val Cys Arg Gly Tyr
785 790 795 800
Leu Ala Arg Lys Ala Phe Ala Lys Lys Gln Gln Gln Leu Ser Ala Leu
805 810 815
Lys Val Leu Gln Arg Asn Cys Ala Ala Tyr Leu Lys Leu Arg His Trp
820 825 830
Gln Trp Trp Arg Val Phe Thr Lys Val Lys Pro Leu Leu Gln Val Thr
835 840 845
Arg Gln Glu Glu Glu Leu Gln Ala Lys Asp Glu Glu Leu Leu Lys Val
850 855 860
Lys Glu Lys Gln Thr Lys Val Glu Gly Glu Leu Glu Glu Met Glu Arg
865 870 875 880
Lys His Gln Gln Leu Leu Glu Glu Lys Asn Ile Leu Ala Glu Gln Leu
885 890 895
Gln Ala Glu Thr Glu Leu Phe Ala Glu Ala Glu Glu Met Arg Ala Arg
900 905 910
Leu Ala Ala Lys Lys Gln Glu Leu Glu Glu Ile Leu His Asp Leu Glu
915 920 925
Ser Arg Val Glu Glu Glu Glu Glu Arg Asn Gln Ile Leu Gln Asn Glu
930 935 940
Lys Lys Lys Met Gln Ala His Ile Gln Asp Leu Glu Glu Gln Leu Asp
945 950 955 960
Glu Glu Glu Gly Ala Arg Gln Lys Leu Gln Leu Glu Lys Val Thr Ala
965 970 975
Glu Ala Lys Ile Lys Lys Met Glu Glu Glu Ile Leu Leu Leu Glu Asp
980 985 990
Gln Asn Ser Lys Phe Ile Lys Glu Lys Lys Leu Met Glu Asp Arg Ile
995 1000 1005
Ala Glu Cys Ser Ser Gln Leu Ala Glu Glu Glu Glu Lys Ala Lys Asn
1010 1015 1020
Leu Ala Lys Ile Arg Asn Lys Gln Glu Val Met Ile Ser Asp Leu Glu
1025 1030 1035 1040
Glu Arg Leu Lys Lys Glu Glu Lys Thr Arg Gln Glu Leu Glu Lys Ala
1045 1050 1055
Lys Arg Lys Leu Asp Gly Glu Thr Thr Asp Leu Gln Asp Gln Ile Ala
1060 1065 1070
Glu Leu Gln Ala Gln Ile Asp Glu Leu Lys Leu Gln Leu Ala Lys Lys
1075 1080 1085
Glu Glu Glu Leu Gln Gly Ala Leu Ala Arg Gly Asp Asp Glu Thr Leu
1090 1095 1100
His Lys Asn Asn Ala Leu Lys Val Val Arg Glu Leu Gln Ala Gln Ile
1105 1110 1115 1120
Ala Glu Leu Gln Glu Asp Phe Glu Ser Glu Lys Ala Ser Arg Asn Lys
1125 1130 1135
Ala Glu Lys Gln Lys Arg Asp Leu Ser Glu Glu Leu Glu Ala Leu Lys
1140 1145 1150
Thr Glu Leu Glu Asp Thr Leu Asp Thr Thr Ala Ala Gln Gln Glu Leu
1155 1160 1165
Arg Thr Lys Arg Glu Gln Glu Val Ala Glu Leu Lys Lys Ala Leu Glu
1170 1175 1180
Glu Glu Thr Lys Asn His Glu Ala Gln Ile Gln Asp Met Arg Gln Arg
1185 1190 1195 1200
His Ala Thr Ala Leu Glu Glu Leu Ser Glu Gln Leu Glu Gln Ala Lys
1205 1210 1215
Arg Phe Lys Ala Asn Leu Glu Lys Asn Lys Gln Gly Leu Glu Thr Asp
1220 1225 1230
Asn Lys Glu Leu Ala Cys Glu Val Lys Val Leu Gln Gln Val Lys Ala
1235 1240 1245
Glu Ser Glu His Lys Arg Lys Lys Leu Asp Ala Gln Val Gln Glu Leu
1250 1255 1260
His Ala Lys Val Ser Glu Gly Asp Arg Leu Arg Val Glu Leu Ala Glu
1265 1270 1275 1280
Lys Ala Ser Lys Leu Gln Asn Glu Leu Asp Asn Val Ser Thr Leu Leu
1285 1290 1295
Glu Glu Ala Glu Lys Lys Gly Ile Lys Phe Ala Lys Asp Ala Ala Ser
1300 1305 1310
Leu Glu Ser Gln Leu Gln Asp Thr Gln Glu Leu Leu Gln Glu Glu Thr
1315 1320 1325
Arg Gln Lys Leu Asn Leu Ser Ser Arg Ile Arg Gln Leu Glu Glu Glu
1330 1335 1340
Lys Asn Ser Leu Gln Glu Gln Gln Glu Glu Glu Glu Glu Ala Arg Lys
1345 1350 1355 1360
Asn Leu Glu Lys Gln Val Leu Ala Leu Gln Ser Gln Leu Ala Asp Thr
1365 1370 1375
Lys Lys Lys Val Asp Asp Asp Leu Gly Thr Ile Glu Ser Leu Glu Glu
1380 1385 1390
Ala Lys Lys Lys Leu Leu Lys Asp Ala Glu Ala Leu Ser Gln Arg Leu
1395 1400 1405
Glu Glu Lys Ala Leu Ala Tyr Asp Lys Leu Glu Lys Thr Lys Asn Arg
1410 1415 1420
Leu Gln Gln Glu Pro Asp Asp Leu Thr Val Asp Leu Asp His Gln Arg
1425 1430 1435 1440
Gln Val Ala Ser Asn Leu Glu Lys Lys Gln Lys Lys Phe Asp Gln Leu
1445 1450 1455
Leu Ala Glu Glu Lys Ser Ile Ser Ala Arg Tyr Ala Glu Glu Arg Asp
1460 1465 1470
Arg Ala Glu Ala Glu Ala Arg Glu Lys Glu Thr Lys Ala Leu Ser Leu
1475 1480 1485
Ala Arg Ala Leu Glu Glu Ala Leu Glu Ala Lys Glu Glu Phe Glu Arg
1490 1495 1500
Gln Asn Lys Gln Leu Arg Ala Asp Met Glu Asp Leu Met Ser Ser Lys
1505 1510 1515 1520
Asp Asp Val Gly Lys Asn Val His Glu Leu Glu Lys Ser Lys Arg Ala
1525 1530 1535
Leu Glu Gln Gln Val Glu Glu Met Arg Thr Gln Leu Glu Glu Leu Glu
1540 1545 1550
Asp Glu Leu Gln Ala Thr Glu Asp Ala Lys Leu Arg Leu Glu Val Asn
1555 1560 1565
Met Gln Ala Met Lys Ala Gln Phe Glu Arg Asp Leu Gln Thr Arg Asp
1570 1575 1580
Glu Gln Asn Glu Glu Lys Lys Arg Leu Leu Ile Lys Gln Val Arg Glu
1585 1590 1595 1600
Leu Glu Ala Glu Leu Glu Asp Glu Arg Lys Gln Arg Ala Leu Ala Val
1605 1610 1615
Ala Ser Lys Lys Lys Met Glu Ile Asp Leu Lys Asp Leu Glu Ala Gln
1620 1625 1630
Ile Glu Ala Ala Asn Lys Ala Arg Asp Glu Val Ile Lys Gln Leu Arg
1635 1640 1645
Lys Leu Gln Ala Gln Met Lys Asp Tyr Gln Arg Glu Leu Glu Glu Ala
1650 1655 1660
Arg Ala Ser Arg Asp Glu Ile Phe Ala Gln Ser Lys Glu Ser Glu Lys
1665 1670 1675 1680
Lys Leu Lys Ser Leu Glu Ala Glu Ile Leu Gln Leu Gln Glu Glu Leu
1685 1690 1695
Ala Ser Ser Glu Arg Ala Arg Arg His Ala Glu Gln Glu Arg Asp Glu
1700 1705 1710
Leu Ala Asp Glu Ile Thr Asn Ser Ala Ser Gly Lys Ser Ala Leu Leu
1715 1720 1725
Asp Glu Lys Arg Arg Leu Glu Ala Arg Ile Ala Gln Leu Glu Glu Glu
1730 1735 1740
Leu Glu Glu Glu Gln Ser Asn Met Glu Leu Leu Asn Asp Arg Phe Arg
1745 1750 1755 1760
Lys Thr Thr Leu Gln Val Asp Thr Leu Asn Ala Glu Leu Ala Ala Glu
1765 1770 1775
Arg Ser Ala Ala Gln Lys Ser Asp Asn Ala Arg Gln Gln Leu Glu Arg
1780 1785 1790
Gln Asn Lys Glu Leu Lys Ala Lys Leu Gln Glu Leu Glu Gly Ala Val
1795 1800 1805
Lys Ser Lys Phe Lys Ala Thr Ile Ser Ala Leu Glu Ala Lys Ile Gly
1810 1815 1820
Gln Leu Glu Glu Gln Leu Glu Gln Glu Ala Lys Glu Arg Ala Ala Ala
1825 1830 1835 1840
Asn Lys Leu Val Arg Arg Thr Glu Lys Lys Leu Lys Glu Ile Phe Met
1845 1850 1855
Gln Val Glu Asp Glu Arg Arg His Ala Asp Gln Tyr Lys Glu Gln Met
1860 1865 1870
Glu Lys Ala Asn Ala Arg Met Lys Gln Leu Lys Arg Gln Leu Glu Glu
1875 1880 1885
Ala Glu Glu Glu Ala Thr Arg Ala Asn Ala Ser Arg Arg Lys Leu Gln
1890 1895 1900
Arg Glu Leu Asp Asp Ala Thr Glu Ala Asn Glu Gly Leu Ser Arg Glu
1905 1910 1915 1920
Val Ser Thr Leu Lys Asn Arg Leu Arg Arg Gly Gly Pro Ile Ser Phe
1925 1930 1935
Ser Ser Ser Arg Ser Gly Arg Arg Gln Leu His Leu Glu Gly Ala Ser
1940 1945 1950
Leu Glu Leu Ser Asp Asp Asp Thr Glu Ser Lys Thr Ser Asp Val Asn
1955 1960 1965
Glu Thr Gln Pro Pro Gln Ser Glu
1970 1975
<210> 11
<211> 222
<212> PRT
<213> Homo sapiens
<400> 11
Met Ala Gln Lys Gly Gln Leu Ser Asp Asp Glu Lys Phe Leu Phe Val
1 5 10 15
Asp Lys Asn Phe Ile Asn Ser Pro Val Ala Gln Ala Asp Trp Ala Ala
20 25 30
Lys Arg Leu Val Trp Val Pro Ser Glu Lys Gln Gly Phe Glu Ala Ala
35 40 45
Ser Ile Lys Glu Glu Lys Gly Asp Glu Val Val Val Glu Leu Val Glu
50 55 60
Asn Gly Lys Lys Val Thr Val Gly Lys Asp Asp Ile Gln Lys Met Asn
65 70 75 80
Pro Pro Lys Phe Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu
85 90 95
Asn Glu Ala Ser Val Leu His Asn Leu Arg Glu Arg Tyr Phe Ser Gly
100 105 110
Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val Val Asn Pro Tyr
115 120 125
Lys His Leu Pro Ile Tyr Ser Glu Lys Ile Val Asp Met Tyr Lys Gly
130 135 140
Lys Lys Arg His Glu Met Pro Pro His Ile Tyr Ala Ile Ala Asp Thr
145 150 155 160
Ala Tyr Arg Ser Met Leu Gln Asp Arg Glu Asp Gln Ser Ile Leu Cys
165 170 175
Thr Gly Glu Ser Gly Ala Trp Lys Thr Glu Asn Thr Lys Lys Val Ile
180 185 190
Gln Tyr Leu Ala Val Val Ala Ser Ser His Lys Gly Lys Lys Asp Thr
195 200 205
Ser Ile Thr Val Ser Gly Ser Ser Gln Ser Glu Ala Met Ile
210 215 220
<210> 12
<211> 208
<212> PRT
<213> Homo sapiens
<400> 12
Met Ala Ser Lys Cys Pro Lys Cys Asp Lys Thr Val Tyr Phe Ala Glu
1 5 10 15
Lys Val Ser Ser Leu Gly Lys Asp Trp His Lys Phe Cys Leu Lys Cys
20 25 30
Glu Arg Cys Ser Lys Thr Leu Thr Pro Gly Gly His Ala Glu His Asp
35 40 45
Gly Lys Pro Phe Cys His Lys Pro Cys Tyr Ala Thr Leu Phe Gly Pro
50 55 60
Lys Gly Val Asn Ile Gly Gly Ala Gly Ser Tyr Ile Tyr Glu Lys Pro
65 70 75 80
Leu Ala Glu Gly Pro Gln Val Thr Gly Pro Ile Glu Val Pro Ala Ala
85 90 95
Arg Ala Glu Glu Arg Lys Ala Ser Gly Pro Pro Lys Gly Pro Ser Arg
100 105 110
Ala Ser Ser Val Thr Thr Phe Thr Gly Glu Pro Asn Thr Cys Pro Arg
115 120 125
Cys Ser Lys Lys Val Tyr Phe Ala Glu Lys Val Thr Ser Leu Gly Lys
130 135 140
Asp Trp His Arg Pro Cys Leu Arg Cys Glu Arg Cys Gly Lys Thr Leu
145 150 155 160
Thr Pro Gly Gly His Ala Glu His Asp Gly Gln Pro Tyr Cys His Lys
165 170 175
Pro Cys Tyr Gly Ile Leu Phe Gly Pro Lys Gly Val Asn Thr Gly Ala
180 185 190
Val Gly Ser Tyr Ile Tyr Asp Arg Asp Pro Glu Gly Lys Val Gln Pro
195 200 205
<210> 13
<211> 1163
<212> PRT
<213> Homo sapiens
<400> 13
Met Thr Arg Thr Arg Ala Ala Leu Leu Leu Phe Thr Ala Leu Ala Thr
1 5 10 15
Ser Leu Gly Phe Asn Leu Asp Thr Glu Glu Leu Thr Ala Phe Arg Val
20 25 30
Asp Ser Ala Gly Phe Gly Asp Ser Val Val Gln Tyr Ala Asn Ser Trp
35 40 45
Val Val Val Gly Ala Pro Gln Lys Ile Thr Ala Ala Asn Gln Thr Gly
50 55 60
Gly Leu Tyr Gln Cys Gly Tyr Ser Thr Gly Ala Cys Glu Pro Ile Gly
65 70 75 80
Leu Gln Val Pro Pro Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu
85 90 95
Ala Ser Thr Thr Ser Pro Ser Gln Leu Leu Ala Cys Gly Pro Thr Val
100 105 110
His His Glu Cys Gly Arg Asn Met Tyr Leu Thr Gly Leu Cys Phe Leu
115 120 125
Leu Gly Pro Thr Gln Leu Thr Gln Arg Leu Pro Val Ser Arg Gln Glu
130 135 140
Cys Pro Arg Gln Glu Gln Asp Ile Val Phe Leu Ile Asp Gly Ser Gly
145 150 155 160
Ser Ile Ser Ser Arg Asn Phe Ala Thr Met Met Asn Phe Val Arg Ala
165 170 175
Val Ile Ser Gln Phe Gln Arg Pro Ser Thr Gln Phe Ser Leu Met Gln
180 185 190
Phe Ser Asn Lys Phe Gln Thr His Leu Thr Phe Glu Glu Phe Arg Arg
195 200 205
Thr Ser Asn Pro Leu Ser Leu Leu Ala Ser Val His Gln Leu Gln Gly
210 215 220
Phe Thr Tyr Thr Ala Thr Ala Ile Gln Asn Val Val His Arg Leu Phe
225 230 235 240
His Ala Ser Tyr Gly Ala Arg Arg Asp Ala Thr Lys Ile Leu Ile Val
245 250 255
Ile Thr Asp Gly Lys Lys Glu Gly Asp Thr Leu Asp Tyr Lys Asp Val
260 265 270
Ile Pro Met Ala Asp Ala Ala Gly Ile Ile Arg Tyr Ala Ile Gly Val
275 280 285
Gly Leu Ala Phe Gln Asn Arg Asn Ser Trp Lys Glu Leu Asn Asp Ile
290 295 300
Ala Ser Lys Pro Ser Gln Glu His Ile Phe Lys Val Glu Asp Phe Asp
305 310 315 320
Ala Leu Lys Asp Ile Gln Thr Gln Leu Arg Glu Lys Ile Phe Pro Ile
325 330 335
Glu Gly Thr Glu Thr Thr Ser Ser Ser Ser Phe Glu Leu Glu Met Ala
340 345 350
Gln Glu Gly Phe Ser Ala Val Phe Thr Pro Asp Gly Pro Val Leu Gly
355 360 365
Ala Val Gly Ser Phe Thr Trp Ser Gly Gly Ala Phe Leu Tyr Pro Pro
370 375 380
Asn Met Ser Pro Thr Phe Ile Asn Met Ser Gln Glu Asn Val Asp Met
385 390 395 400
Arg Asp Ser Tyr Leu Gly Tyr Ser Thr Glu Leu Ala Leu Trp Lys Gly
405 410 415
Val Gln Ser Leu Val Leu Gly Ala Pro Arg Tyr Gln His Thr Gly Lys
420 425 430
Ala Val Ile Phe Thr Gln Val Ser Arg Gln Trp Arg Met Lys Ala Glu
435 440 445
Val Thr Gly Thr Gln Ile Gly Ser Tyr Phe Gly Pro Ser Leu Cys Ser
450 455 460
Val Asp Val Asp Ser Asp Gly Ser Thr Asp Leu Val Leu Ile Gly Pro
465 470 475 480
Pro His Tyr Tyr Glu Gln Thr Arg Gly Ala Gln Val Ser Val Cys Pro
485 490 495
Leu Pro Arg Gly Trp Arg Arg Trp Trp Cys Asp Ala Val Leu Tyr Gly
500 505 510
Glu Gln Gly His Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val Leu
515 520 525
Gly Asp Val Asn Gly Asp Lys Leu Thr Asp Val Val Ile Gly Ala Pro
530 535 540
Gly Glu Glu Glu Asn Arg Gly Ala Val Tyr Leu Phe His Gly Val Leu
545 550 555 560
Gly Pro Ser Ile Ser Pro Ser His Ser Gln Arg Ile Ala Gly Ser Gln
565 570 575
Leu Ser Ser Arg Leu Gln Tyr Phe Gly Gln Ala Leu Ser Gly Gly Gln
580 585 590
Asp Leu Thr Gln Asp Gly Leu Val Asp Leu Ala Val Gly Ala Arg Gly
595 600 605
Gln Val Leu Leu Leu Arg Thr Arg Pro Val Leu Trp Val Gly Val Ser
610 615 620
Met Gln Phe Ile Pro Ala Glu Ile Pro Arg Ser Ala Phe Glu Cys Arg
625 630 635 640
Glu Gln Val Val Ser Glu Gln Thr Leu Val Gln Ser Asn Ile Cys Leu
645 650 655
Tyr Ile Asp Lys Arg Ser Lys Asn Leu Leu Gly Ser Arg Asp Leu Gln
660 665 670
Ser Ser Val Thr Leu Asp Leu Ala Leu Asp Pro Gly Arg Leu Ser Pro
675 680 685
Arg Ala Thr Phe Gln Glu Thr Lys Asn Arg Ser Leu Ser Arg Val Arg
690 695 700
Val Leu Gly Leu Lys Ala His Cys Glu Asn Phe Asn Leu Leu Leu Pro
705 710 715 720
Ser Cys Val Glu Asp Ser Val Thr Pro Ile Thr Leu Arg Leu Asn Phe
725 730 735
Thr Leu Val Gly Lys Pro Leu Leu Ala Phe Arg Asn Leu Arg Pro Met
740 745 750
Leu Ala Ala Asp Ala Gln Arg Tyr Phe Thr Ala Ser Leu Pro Phe Glu
755 760 765
Lys Asn Cys Gly Ala Asp His Ile Cys Gln Asp Asn Leu Gly Ile Ser
770 775 780
Phe Ser Phe Pro Gly Leu Lys Ser Leu Leu Val Gly Ser Asn Leu Glu
785 790 795 800
Leu Asn Ala Glu Val Met Val Trp Asn Asp Gly Glu Asp Ser Tyr Gly
805 810 815
Thr Thr Ile Thr Phe Ser His Pro Ala Gly Leu Ser Tyr Arg Tyr Val
820 825 830
Ala Glu Gly Gln Lys Gln Gly Gln Leu Arg Ser Leu His Leu Thr Cys
835 840 845
Asp Ser Ala Pro Val Gly Ser Gln Gly Thr Trp Ser Thr Ser Cys Arg
850 855 860
Ile Asn His Leu Ile Phe Arg Gly Gly Ala Gln Ile Thr Phe Leu Ala
865 870 875 880
Thr Phe Asp Val Ser Pro Lys Ala Val Leu Gly Asp Arg Leu Leu Leu
885 890 895
Thr Ala Asn Val Ser Ser Glu Asn Asn Thr Pro Arg Thr Ser Lys Thr
900 905 910
Thr Phe Gln Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Thr Val Val
915 920 925
Ser Ser His Glu Gln Phe Thr Lys Tyr Leu Asn Phe Ser Glu Ser Glu
930 935 940
Glu Lys Glu Ser His Val Ala Met His Arg Tyr Gln Val Asn Asn Leu
945 950 955 960
Gly Gln Arg Asp Leu Pro Val Ser Ile Asn Phe Trp Val Pro Val Glu
965 970 975
Leu Asn Gln Glu Ala Val Trp Met Asp Val Glu Val Ser Leu Pro Gln
980 985 990
Asn Pro Ser Leu Arg Cys Ser Ser Glu Lys Ile Ala Gly Pro Ala Ser
995 1000 1005
Asp Phe Leu Ala His Ile Gln Lys Asn Pro Val Leu Asp Cys Ser Ile
1010 1015 1020
Ala Gly Cys Leu Arg Phe Arg Cys Asp Val Pro Ser Phe Ser Val Gln
1025 1030 1035 1040
Glu Glu Leu Asp Phe Thr Leu Lys Gly Asn Leu Ser Phe Gly Trp Val
1045 1050 1055
Arg Gln Ile Leu Gln Lys Lys Val Ser Val Val Ser Val Ala Glu Ile
1060 1065 1070
Thr Phe Asp Thr Ser Val Tyr Ser Gln Leu Pro Gly Gln Glu Ala Phe
1075 1080 1085
Met Arg Ala Gln Thr Thr Thr Val Leu Glu Lys Tyr Lys Val His Asn
1090 1095 1100
Pro Thr Pro Leu Ile Val Gly Ser Ser Ile Gly Gly Leu Leu Leu Leu
1105 1110 1115 1120
Ala Leu Ile Thr Ala Val Leu Tyr Lys Val Gly Phe Phe Lys Arg Gln
1125 1130 1135
Tyr Lys Glu Met Met Glu Glu Ala Asn Gly Gln Ile Ala Pro Glu Asn
1140 1145 1150
Gly Thr Gln Thr Pro Ser Pro Pro Ser Glu Lys
1155 1160
Claims (18)
- TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코팅되는 단백질의 발현 수준을 측정하는 제제를 포함하는, 암의 예후 예측용 조성물.
- 제 1항에 있어서,
상기 유전자의 발현 수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택된 1 종 이상을 포함하는, 조성물. - 제 1항에 있어서,
상기 단백질의 발현 수준을 측정하는 제제는 상기 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA (peptide nucleic acid) 및 앱타머 (aptamer)로 이루어진 군에서 선택된 1 종 이상을 포함하는, 조성물. - 제 1항에 있어서,
상기 암은 예후가 좋지 않은 악성 암으로서 줄기세포의 특성을 갖는 암인, 조성물. - 제 1항에 있어서,
상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 조성물. - 제 1항 내지 제 5항 중 어느 한 항의 조성물을 포함하는, 암의 예후 예측용 키트.
- 제 6항에 있어서,
상기 키트는 RT-PCR 키트, DNA 칩 키트, ELISA 키트, 단백질 칩 키트, 래피드 (rapid) 키트 또는 MRM (Multiple reaction monitoring) 키트인, 키트. - 암이 발병하였거나 발병 가능성이 높은 목적하는 개체로부터 분리된 생물학적 시료에서 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준을 측정하는 단계를 포함하는, 암의 예후 예측을 위한 정보 제공 방법.
- 제 8항에 있어서,
상기 유전자의 발현 수준을 측정하는 단계는 상기 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택된 1 종 이상을 포함하는 제제를 이용하는 것인, 방법. - 제 8항에 있어서,
상기 단백질의 발현 수준을 측정하는 단계는 상기 단백질에 특이적으로 결합하는 항체, 올리고펩타이드, 리간드, PNA (peptide nucleic acid) 및 앱타머 (aptamer)로 이루어진 군에서 선택된 1 종 이상을 포함하는 제제를 이용하는 것인, 방법. - 제 8항에 있어서,
상기 발현 수준을 측정하는 단계 시 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 2 개의 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준을 측정하고,
측정된 상기 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준의 로그 강도(log intensity)의 평균 값인 SMC(smooth muscle cell) 스코어 값을 계산하는 단계를 더 포함하는, 방법. - 제 11항에 있어서,
계산된 SMC 스코어 값이 5 내지 15의 값 이상인 경우 목적하는 개체의 치료 예후가 좋지 않거나 생존율이 낮거나 생존 기간이 짧을 것으로 예측하는 단계;를 추가로 포함하는, 방법. - 제 8항에 있어서,
계산된 SMC 스코어 값이 5 내지 15의 값 미만인 경우 목적하는 개체의 치료 예후가 좋거나 생존율이 높거나 생존 기간이 길 것으로 예측하는 단계;를 추가로 포함하는, 방법. - 제 8항에 있어서,
상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 방법. - (a) 목적하는 개체로부터 얻어진 생물학적 시료에 대하여 TAGLN, CALD1, MYL9, MYLK, ACTA2, TPM2, CNN1, MSN, CNN3, MYH10, MYH11, CRIP2 및 ITGA5로 이루어진 군에서 선택된 적어도 하나의 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준을 측정하는 측정부; 및
(b) 상기 측정부에서 측정된 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준으로부터 SMC 스코어 값을 계산하는 연산부;를 포함하는 암의 예후 진단기기. - 제 15항에 있어서,
상기 기기는 연산부에서 예측된 목적하는 개체의 암의 예후를 출력하는 출력부를 추가로 더 포함하는, 진단기기. - 제 15항에 있어서,
상기 암은 예후가 좋지 않은 악성 암으로서 줄기세포의 특성을 갖는 암인, 진단기기. - 제 15항에 있어서,
상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 진단기기.
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