KR20230175021A - A novel biomarker that can predict the diagnosis and prognosis of bladder cancer in serum or urine - Google Patents
A novel biomarker that can predict the diagnosis and prognosis of bladder cancer in serum or urine Download PDFInfo
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Abstract
본 발명은 혈청 또는 소변에서 방광암의 진단, 전이 및 예후를 예측할 수 있는 신규한 바이오마커에 관한 것으로서, 본 발명의 방광암 바이오마커인 STC1은, 방광암 환자에서 고발현 되면, 환자의 불량한 예후와 관계된 것을 확인하였다. 또한, STC1의 발현이 증가할수록, 방광암 세포의 증식, 침윤 및 전이가 증가하는 것을 확인하였다. 또한, STC1이 방광암 환자의 혈청 또는 소변에서 검출되고, 환자의 임상 병기에 따른 발현 차이를 확인하여, 효과적으로 방광암의 진단 및 예후 예측에 이용될 수 있는 것을 확인하였다.The present invention relates to a novel biomarker that can predict the diagnosis, metastasis, and prognosis of bladder cancer in serum or urine. STC1, a bladder cancer biomarker of the present invention, is associated with a poor prognosis of patients when expressed at high levels in bladder cancer patients. Confirmed. In addition, it was confirmed that as the expression of STC1 increased, the proliferation, invasion, and metastasis of bladder cancer cells increased. In addition, it was confirmed that STC1 was detected in the serum or urine of bladder cancer patients, and that expression differences depending on the patient's clinical stage were confirmed, and that it can be effectively used to diagnose and predict the prognosis of bladder cancer.
Description
본 발명은 혈청 또는 소변에서 방광암의 진단 및 예후를 예측할 수 있는 신규한 바이오마커에 관한 것이다.The present invention relates to a novel biomarker that can diagnose and predict the prognosis of bladder cancer in serum or urine.
방광암은 비뇨기계 암 중에서는 가장 흔한 암이며, 사회가 점차 고령화되어감에 따라 방광암으로 진단 받는 환자들의 수는 증가되고 있다. 방광암 환자들이 진단 받을 시, 약 70%가 방광의 근육층으로 침범하지 않은 표재성 방광암으로 진단 받으며, 치료 시 5년 생존율은 70%에 달한다. 그러나 그 중 50% 이상이 재발 시 표재성 방광암으로 나타날 뿐만 아니라 일부 환자들은 근육층까지 침범한 침윤성 방광암 또는 전이성 방광암으로 나타난다. 방광암 환자들의 성공적인 치료에 가장 큰 문제점은 항암제 내성과 빈번한 재발이다. 이러한 이유로 방광암 환자들은 지속적인 모니터링이 필요하기 때문에 고비용과 검사 후 부작용이 있는 방광경 검사를 빈번하게 시행한다. 또한, 화학 항암제 요법과 같은 치료에도 불구하고 방광암 환자의 재발 문제는 계속해서 삶의 질을 저하시키고 재정적 고통을 유발한다. 따라서 방광암 진단이 보다 안전하고 간편하며, 환자의 항암제 내성 및 예후 예측까지 가능한 바이오마커 개발이 필수적이다.Bladder cancer is the most common cancer among urinary system cancers, and as society gradually ages, the number of patients diagnosed with bladder cancer is increasing. When patients with bladder cancer are diagnosed, approximately 70% are diagnosed with superficial bladder cancer that has not invaded the muscle layer of the bladder, and the 5-year survival rate with treatment reaches 70%. However, more than 50% of cases of recurrence not only manifest as superficial bladder cancer, but some patients also develop invasive bladder cancer or metastatic bladder cancer that has invaded the muscle layer. The biggest problem in successful treatment of bladder cancer patients is anticancer drug resistance and frequent recurrence. For this reason, bladder cancer patients frequently undergo cystoscopy, which is expensive and has side effects after the test because it requires continuous monitoring. Additionally, despite treatments such as chemotherapy, recurrence problems in bladder cancer patients continue to reduce quality of life and cause financial distress. Therefore, it is essential to develop a biomarker that makes bladder cancer diagnosis safer and easier and can even predict the patient's anticancer drug resistance and prognosis.
본 발명의 목적은 혈청 또는 소변에서 방광암 진단, 전이 또는 예후 예측을 위한 STC1(Stanniocalcin-1) 바이오마커를 제공하는 것이다.The purpose of the present invention is to provide a STC1 (Stanniocalcin-1) biomarker for diagnosing, metastasizing, or predicting prognosis of bladder cancer in serum or urine.
본 발명의 다른 목적은 STC1(Stanniocalcin-1)의 발현수준을 측정할 수 있는 제제를 포함하는, 방광암(bladder cancer)의 진단, 전이 또는 예후 예측용 바이오마커 조성물을 제공하는 것이다.Another object of the present invention is to provide a biomarker composition for diagnosing, metastasizing, or predicting prognosis of bladder cancer, including an agent capable of measuring the expression level of STC1 (Stanniocalcin-1).
본 발명의 또 다른 목적은 상기의 조성물을 포함하는 방광암(bladder cancer)의 진단, 전이 또는 예후 예측용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for diagnosing, metastasizing, or predicting prognosis of bladder cancer, comprising the above composition.
본 발명의 또 다른 목적은 개체로부터 생물학적 시료를 분리하는 단계;Another object of the present invention is to isolate a biological sample from an individual;
상기 분리된 생물학적 시료에서 STC1(Stanniocalcin-1)의 발현수준을 측정하는 단계; 및Measuring the expression level of STC1 (Stanniocalcin-1) in the separated biological sample; and
상기 STC1의 발현 수준을 대조군의 기준치와 비교하는 단계;를 포함하는 방광암(bladder cancer)의 진단, 전이 또는 예후 예측을 위한 정보 제공 방법을 제공하는 것이다.To provide a method of providing information for diagnosis, metastasis, or prognosis of bladder cancer, including comparing the expression level of STC1 with a reference value of a control group.
본 발명의 또 다른 목적은 개체부터 생물학적 시료를 분리하는 단계;Another object of the present invention is to separate a biological sample from an individual;
상기 분리된 생물학적 시료에 후보 물질을 처리하는 단계;Processing the separated biological sample with a candidate substance;
상기 후보물질이 처리된 생물학적 시료에서 STC1(Stanniocalcin-1)의 발현 수준을 측정하는 단계; 및Measuring the expression level of STC1 (Stanniocalcin-1) in the biological sample treated with the candidate material; and
상기 STC1의 발현 수준을 대조군의 기준치와 비교하는 단계;를 포함하는 방광암(bladder cancer) 치료제의 스크리닝 방법을 제공하는 것이다.To provide a screening method for a treatment for bladder cancer, comprising comparing the expression level of STC1 with a reference value of a control group.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 혈청 또는 소변에서 방광암 진단, 전이 또는 예후 예측을 위한 STC1(Stanniocalcin-1) 바이오마커를 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a STC1 (Stanniocalcin-1) biomarker for diagnosing, metastasizing, or predicting prognosis of bladder cancer in serum or urine.
또한, 본 발명은 STC1(Stanniocalcin-1)의 발현수준을 측정할 수 있는 제제를 포함하는, 방광암(bladder cancer)의 진단, 전이 또는 예후 예측용 바이오마커 조성물을 제공한다.Additionally, the present invention provides a biomarker composition for diagnosing, metastasizing, or predicting prognosis of bladder cancer, including an agent capable of measuring the expression level of STC1 (Stanniocalcin-1).
또한, 본 발명은 상기의 조성물을 포함하는 방광암(bladder cancer)의 진단, 전이 또는 예후 예측용 키트를 제공한다.Additionally, the present invention provides a kit for diagnosing, metastasizing, or predicting prognosis of bladder cancer, comprising the composition described above.
또한, 본 발명은 개체로부터 생물학적 시료를 분리하는 단계;Additionally, the present invention includes the steps of isolating a biological sample from an individual;
상기 분리된 생물학적 시료에서 STC1(Stanniocalcin-1)의 발현수준을 측정하는 단계; 및Measuring the expression level of STC1 (Stanniocalcin-1) in the separated biological sample; and
상기 STC1의 발현 수준을 대조군의 기준치와 비교하는 단계;를 포함하는 방광암(bladder cancer)의 진단, 전이 또는 예후 예측을 위한 정보 제공 방법을 제공한다.It provides a method of providing information for diagnosis, metastasis, or prognosis prediction of bladder cancer, including the step of comparing the expression level of STC1 with a reference value of a control group.
또한, 본 발명은 개체부터 생물학적 시료를 분리하는 단계;In addition, the present invention includes the steps of separating a biological sample from an individual;
상기 분리된 생물학적 시료에 후보 물질을 처리하는 단계;Processing the separated biological sample with a candidate substance;
상기 후보물질이 처리된 생물학적 시료에서 STC1(Stanniocalcin-1)의 발현 수준을 측정하는 단계; 및Measuring the expression level of STC1 (Stanniocalcin-1) in the biological sample treated with the candidate material; and
상기 STC1의 발현 수준을 대조군의 기준치와 비교하는 단계;를 포함하는 방광암(bladder cancer) 치료제의 스크리닝 방법을 제공한다.It provides a screening method for a treatment for bladder cancer, comprising comparing the expression level of STC1 with a reference value of a control group.
본 발명의 방광암 바이오마커인 STC1은, 방광암 환자에서 고발현 되면, 환자의 불량한 예후와 관계된 것을 확인하였다. 또한, STC1의 발현이 증가할수록, 방광암 세포의 증식, 침윤 및 전이가 증가하는 것을 확인하였다. 또한, STC1이 방광암 환자의 혈청 또는 소변에서 검출되고, 환자의 임상 병기에 따른 발현 차이를 확인하여, 효과적으로 방광암의 진단 및 예후 예측에 이용될 수 있는 것을 확인하여, 관련 산업에 유용하게 이용할 수 있다.It was confirmed that STC1, the bladder cancer biomarker of the present invention, is associated with poor prognosis of patients when highly expressed in bladder cancer patients. In addition, it was confirmed that as the expression of STC1 increased, the proliferation, invasion, and metastasis of bladder cancer cells increased. In addition, STC1 was detected in the serum or urine of bladder cancer patients, and expression differences according to the patient's clinical stage were confirmed, confirming that it can be effectively used for diagnosing and predicting prognosis of bladder cancer, making it useful in related industries. .
도 1은 T24(P0) 및 GRC1-P15(P15) 세포의 조건 배지의 확인 및 정량 비교를 나타낸 것이다. (a) P0 및 P15 세포에 혈청이 제외된 배지로부터 LC-MS/MS을 사용하여 분비 단백질 확인을 위한 분비체 분석 과정을 나타낸 개략도이다. 3개 독립된 세포 조건 배지에서 P0 및 P15 세포의 조건 배지에 대한 다양한 분비 단백질 발현을 나타내는 히트맵이다. 오른쪽 그림은 P0 및 P15 세포 배양액으로부터 유래한 조정 배지에서 STC1 단백질 발현 및 ponceau 염색 결과를 나타낸 것이다. (b) P0 및 P15 세포 조건 배지에서 확인된 단백질의 수를 나타낸 벤다이어그램이다. P0 세포에 비해 P15 세포의 조정 배지에서 가장 유의미하게 변한 상위 13개 표준 경로를 나타낸 것이다. (c) P0 및 P15 세포의 조건 배지 사이에서, P15에서 특이적으로 확인된 386개의 단백질들을 위치 및 과발현에 따라 요약하여 총 27개의 단백질로 표시한 것이다.
도 2는 STC1이 방광암에서 좋지 않은 예후를 예측하는 인자임을 나타낸 것이다. (a) 3개 방광암 코호트에서 STC1 발현 수치의 Boxplot이다. (b) 3개의 방광암 코호트에 대한 설명 표이다. (c) STC1과 연관된 유전자들의 발현 프로파일을 나타낸 것이다. 발현 수준이 STC1과 밀접하게 연관된 총 367개 유전자를 클러스터 분석을 위해 선별하였다(Pearson correlation test, P < 0.001, |r| > 0.4). 해당 환자는 STC1 발현이 높은 high 및 낮은 low, 두 그룹으로 나누었다. 한국인 방광암 환자 코호트(GSE13507)에서 STC1 발현에 따른 무진행(Progression-free) 및 암 특이(cancer specific) 생존을 나타낸 것이다. (d) DAVID software를 사용하여 결정한 Gene Ontology 기반 생물학적 기능을 나타낸 것이다. 유의성의 한계값은 P < 0.001 및 FDR < 0.25이다. 또한 오른쪽 도표는 STC1과 밀접하게 연관되는 발현 수준을 갖는 총 367개 유전자 중 21개 유전자를 나타낸 것이다. (e) STC1-연관 유전자의 유전자 발현 프로파일을 나타낸 것이다. 한국인 방광암 환자 코호트(GSE13507)로부터 수득한 367개 유전자 중, Lund 코호트에 공통적으로 포함되는 345개 유전자를 분석하였다. 환자는 STC1 high 및 STC1 low의 두 그룹으로 나누었으며, 그래프는 Lund 코호트에서 STC1 발현에 따른 무진행(Progression-free) 및 암 특이(cancer specific) 생존을 나타낸 것이다. (f) STC1-유전자의 유전자 발현 프로파일을 나타냈으며, 한국인 방광암 환자 코호트(GSE13507)로부터 수득한 367개 유전자 중, 연세 코호트와 공통으로 포함하는 308개 유전자를 분석하였다. 환자는 STC1 high 및 STC1 low의 두 그룹으로 나누었으며, 그래프는 연세 코호트에서 STC1 발현에 따른 암 특이(cancer specific) 생존을 나타낸 것이다.
도 3은 STC1의 발현조절이 방광암 세포의 성장 및 증식을 조절한다는 내용을 나타낸 것이다. (a) 및 (b)는 STC1 과발현 및 녹다운 세포 각각에서 qRT-PCR 및 웨스턴 블롯 결과로서, STC1 mRNA 및 단백질 발현 수준을 나타낸 것이다. (b) STC1 과발현 벡터(pSTC1) #1, #2, 및 #3을 형질주입한 세포주들과 STC1 녹다운 siRNA (siSTC1) #1, #2, 및 #3을 형질주입한 세포주들의 생존률을 MTT 실험을 통해 수행한 결과이다. (c) 3가지 후보군들 중에서 가장 STC1 과발현 및 녹다운 효율이 좋은 pSTC1#1 과 siSTC#2를 형질주입한 세포의 클론원성 검정 결과이다. 데이터는 적어도 3회의 독립 실험으로부터의 평균 ± SD로 나타냈다. **, P < 0.01; ***, P < 0.001.
도 4는 STC1가 방광암 세포의 증식, 침습, 및 이동을 촉진함을 나타낸다. (a) 대조군 세포와 STC1 overexpressing (STC1 OE), 즉 STC1 과발현 세포주에서 STC1 mRNA 및 단백질의 발현 수준을 나타낸 결과이다. (b) 왼쪽 그래프는 대조군 세포와 비교하여 STC1 과발현 세포에서 세포의 생존률을 확인한 MTT 검정 결과이다. 오른쪽은 대조군 세포와 비교하여 STC1 과발현 세포의 종양형성에 대한 효과를 나타내는 클론원성 검정 결과이다. (c) 대조군 세포와 비교하여 STC1 과발현 세포의 트랜스웰을 이용한 세포 침습 및 이동 검정 결과이다. **, P < 0.01; ***, P < 0.001.
도 5은 한국인 방광암 환자 데이터세트에서 (a) STC1과 EMT-관련 유전자 (11 genes, VIM, ZEB1, ZEB2, SNAI1, TWIST1, TWIST2, MMP1, MMP3, MMP9, NCAD, 및 CD44)의 양의 상관관계 연관성 및 (b) STC1과 mesenchymal-epithelial transition (MET)-관련 유전자 (3 genes, SDC1, SDC2, 및 ECAD)의 음의 상관관계 연관성을 나타낸 것이다.
도 6은 방광암 세포에서 STC1 발현이 EMT-관련 유전자들의 발현을 증가시킴을 나타낸 결과이다. (a) 대조군 세포(pcDNA)와 비교하여 STC1 과발현 벡터(pSTC1)를 형질주입한 세포에서 발현된 EMT 및 MET-관련 유전자들 (MMP1, MMP2, MMP9, VIM, SNAIL, SLUG, ZEB1, ZEB2, TWIST, NCAD, SDC1, SDC2, 및 ECAD)의 mRNA 수준을나타낸 것이다. (b) 그 중 확인된 EMT, MET 관련 유전자들 (MMP1, MMP2, MMP9, NCAD, VIM, SNAIL, 및 ECAD)의 단백질 발현을 대조군 세포(pcDNA)와 비교한 웨스턴 블롯 분석 결과이다. ns, no significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
도 7은 STC1 과발현이 생체 내에서 종양의 성장을 촉진함을 나타낸 것이다. (a) 마우스 종양형성 실험 공정을 나타낸 공정도이다. 피하 주입은 마우스 환경 적응 기간 후 7일째에 이루어졌으며 무작위로 2개 그룹(그룹 당 n = 7; Con (Control, 대조군 세포), STC1 OE (STC1 overexpressing, STC1 과발현 세포)으로 나누었다. 마우스 옆구리에 대조군 및 STC1 과발현 세포 (1×106 세포)를 주입하고 매주 체중을 측정했다. (b) 마지막 측정 후 마우스를 희생시켜 종양 조직을 회수하였다. 35일간 캘리퍼스로 종양 부피를 측정한 결과를 나타낸 것이다. 오른쪽 그림은 마우스로부터 수술적으로 제거한 종양의 이미지이다. (c) STC1 과발현 세포 주입에 의한 마우스 종양 조직에서 면역조직화학적으로 H&E 및 STC1 발현을 확인한 염색 사진 결과이다. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
도 8은 STC1 과발현이 생체 내에서 종양의 폐 전이를 촉진함을 나타낸 것이다. (a) 마우스 종양 전이 실험 공정을 나타낸 공정도이다. 정맥 내 주입은 마우스 환경 적응 기간 후 7일째에 이루어졌으며 무작위로 2개 그룹(그룹 당 n = 7; Con (Control, 대조군 세포), STC1 OE (STC1 overexpressing, STC1 과발현 세포)으로 나누었다. 마우스의 꼬리 정맥으로 대조군 및 STC1 과발현 세포 (5×105 세포)를 주입하고 매주 체중을 측정했다. (b) 대조군 및 STC1 과발현 세포 주입으로 형성된 폐 전이가 확인된 마우스 폐 조직의 이미지와 개수를 확인한 결과이다. 오른쪽 그림은 H&E, Ki67 및 STC1을 면역조직화학염색법을 통해 확인한 결과이다. (c) 방광암 환자 Grade 1, 2, 및 3 단계의 방광암 조직의 H&E 염색 및 STC1 발현 정도를 면역조직염색화학법에 의해 나타난 결과이다. *, P < 0.05.
도 9는 STC1 과발현 세포의 조정 배지를 처리한 방광암 세포의 이동 및 침습이 촉진되고, 방광암 세포에서 분비성 STC1 단백질 발현을 확인한 결과이다. (a) 왼쪽은 대조군 및 STC1 과발현 세포의 조정배지 속 STC1 단백질을 확인한 결과이다. 오른쪽은 대조군 및 STC1 과발현 세포의 조정배지를 처리한 방광암 세포의 MTT 검정 결과를 나타낸 것이다. (b) 대조군 및 STC1 과발현 세포의 조정배지를 처리한 방광암 세포의 콜로니 형성 능력이 개선됨을 나타낸 것이다. (c) 대조군 및 STC1 과발현 세포의 조정배지를 처리한 방광암 세포의 침습 및 이동 검정 결과를 나타낸 것이다. (d) 방광암 세포가 배양된 플레이트에 상처를 내고, 대조군 및 STC1 과발현 세포의 조정배지를 처리하여 24시간 후에 회복된 상처 영역(%) 이미지를 나타낸 것이다. 오른쪽은 대조군 및 STC1 과발현 세포의 조정배지를 처리한 방광암 세포의 EMT 마커의 웨스턴 블롯 분석 결과이다. (e) P0 및 P15 세포, 대조군 및 STC1 과발현 벡터 형질주입한 세포, 대조군 및 STC1 과발현 세포에서 분비성 STC1 단백질 발현을 ELISA 검정 결과이다. (f) 도 10에서 확인한 마우스 혈청에서 분비성 STC1 단백질을 ELISA 검정을 통해 확인한 결과이다. **, P < 0.01; ***, P < 0.001.
도 10은 방광암 세포에서 분비성 STC1 단백질이 세포의 증식 및 전이 능력을 증가시킴을 나타낸 것이다. (a) recombinant human STC1 (rhSTC1)을 배지에 처리한 P0 및 P15에서 세포의 증식을 MTT 검정을 통해 확인한 결과이다. (b) rhSTC1을 배지에 첨가하여 P0 세포에서 콜로니 형성, 세포의 침습 및 이동 능력을 확인한 결과이다. (c) rhSTC1에 의해 증가된 방광암 세포에 STC1 항체 (STC1 antibody, ab)에 의해 중화되는 침습 및 이동 능력을 확인한 결과이다. 오른쪽 그림은 rhSTC1에 의한 p-FAK 활성화를 확인한 웨스턴 블롯 결과이다. *, P < 0.05; **, P < 0.01
도 11은 방광암 환자의 혈청 및 소변 샘플에서 분비성 STC1 단백질을 확인함으로서 바이오마커로서 가능성을 확인한 결과이다. (a) 건강한 개체 및 방광암 환자의 혈청 샘플에서 ELISA 검정을 통해 확인한 STC1 농도를 나타낸 것이다. 데이터를 포함하는 스캐터 플롯은 ELISA 검정에 의해 혈청 내 STC1의 수준을 측정한 범위 및 분포를 나타내기 위해 가리키는 것이다. 방광암 환자를 Grade, stage, recurrence로 나누어 비교한 결과를 나타낸다. (b) 건강한 개체 및 방광암 환자의 소변 샘플에서 ELISA 검정을 통해 확인한 STC1 농도를 나타낸 것이다. 데이터를 포함하는 스캐터 플롯은 ELISA 검정에 의해 소변 내 STC1의 수준을 측정한 범위 및 분포를 나타내기 위해 가리키는 것이다. 방광암 환자를 Grade, stage, recurrence로 나누어 비교한 결과를 나타낸다. ***, P < 0.001.Figure 1 shows the identification and quantitative comparison of conditioned media for T24 (P0) and GRC1-P15 (P15) cells. (a) Schematic diagram showing the secretome analysis process for identifying secreted proteins using LC-MS/MS from serum-excluded medium in P0 and P15 cells. Heatmap showing expression of various secreted proteins for conditioned media of P0 and P15 cells in three independent cell conditioned media. The picture on the right shows STC1 protein expression and ponceau staining results in conditioned media derived from P0 and P15 cell cultures. (b) Venn diagram showing the number of proteins identified in P0 and P15 cell condition medium. The top 13 standard pathways that changed most significantly in the conditioned medium of P15 cells compared to P0 cells are shown. (c) Between the conditioned media of P0 and P15 cells, 386 proteins specifically identified in P15 were summarized according to location and overexpression, resulting in a total of 27 proteins.
Figure 2 shows that STC1 is a factor predicting poor prognosis in bladder cancer. (a) Boxplot of STC1 expression levels in three bladder cancer cohorts. (b) This is a descriptive table for the three bladder cancer cohorts. (c) Shows the expression profile of genes associated with STC1. A total of 367 genes whose expression levels were closely related to STC1 were selected for cluster analysis (Pearson correlation test, P < 0.001, |r| > 0.4). The patients were divided into two groups, high and low STC1 expression. Progression-free and cancer-specific survival according to STC1 expression was shown in the Korean bladder cancer patient cohort (GSE13507). (d) Gene Ontology-based biological function determined using DAVID software. The thresholds for significance are P < 0.001 and FDR < 0.25. Additionally, the diagram on the right shows 21 genes out of a total of 367 genes with expression levels closely related to STC1. (e) Shows the gene expression profile of STC1-related genes. Among 367 genes obtained from the Korean bladder cancer patient cohort (GSE13507), 345 genes commonly included in the Lund cohort were analyzed. Patients were divided into two groups, STC1 high and STC1 low, and the graph shows progression-free and cancer specific survival according to STC1 expression in the Lund cohort. (f) The gene expression profile of the STC1-gene is shown, and among 367 genes obtained from the Korean bladder cancer patient cohort (GSE13507), 308 genes included in common with the Yonsei cohort were analyzed. Patients were divided into two groups, STC1 high and STC1 low, and the graph shows cancer specific survival according to STC1 expression in the age cohort.
Figure 3 shows that regulating the expression of STC1 regulates the growth and proliferation of bladder cancer cells. (a) and (b) show the qRT-PCR and Western blot results of STC1 mRNA and protein expression levels in STC1 overexpressing and knockdown cells, respectively. (b) MTT experiment on the survival rates of cell lines transfected with STC1 overexpression vector (pSTC1) #1, #2, and #3 and cell lines transfected with STC1 knockdown siRNA (siSTC1) #1, #2, and #3 This is the result of the process. (c) Clonogenic test results of cells transfected with pSTC1#1 and siSTC#2, which have the best STC1 overexpression and knockdown efficiency among the three candidates. Data are expressed as mean ± SD from at least three independent experiments. **, P <0.01; ***, P < 0.001.
Figure 4 shows that STC1 promotes proliferation, invasion, and migration of bladder cancer cells. (a) Results showing the expression levels of STC1 mRNA and protein in control cells and STC1 overexpressing (STC1 OE), that is, STC1 overexpressing cell line. (b) The graph on the left shows the results of the MTT assay confirming the cell survival rate in STC1 overexpressing cells compared to control cells. On the right is the result of a clonogenic assay showing the effect on tumorigenesis of STC1 overexpressing cells compared to control cells. (c) Results of cell invasion and migration assay using transwell of STC1 overexpressing cells compared to control cells. **, P <0.01; ***, P < 0.001.
Figure 5 shows (a) positive correlation between STC1 and EMT-related genes (11 genes, VIM, ZEB1, ZEB2, SNAI1, TWIST1, TWIST2, MMP1, MMP3, MMP9, NCAD, and CD44) in the Korean bladder cancer patient dataset. (b) negative correlation between STC1 and mesenchymal-epithelial transition (MET)-related genes (3 genes, SDC1, SDC2, and ECAD).
Figure 6 shows results showing that STC1 expression increases the expression of EMT-related genes in bladder cancer cells. (a) EMT and MET-related genes (MMP1, MMP2, MMP9, VIM, SNAIL, SLUG, ZEB1, ZEB2, TWIST) expressed in cells transfected with STC1 overexpression vector (pSTC1) compared to control cells (pcDNA). , NCAD, SDC1, SDC2, and ECAD) mRNA levels are shown. (b) This is the result of Western blot analysis comparing the protein expression of the identified EMT and MET related genes (MMP1, MMP2, MMP9, NCAD, VIM, SNAIL, and ECAD) with control cells (pcDNA). ns, no significant; *, P <0.05; **, P <0.01; ***, P < 0.001.
Figure 7 shows that STC1 overexpression promotes tumor growth in vivo. (a) This is a process diagram showing the mouse tumor formation experiment process. Subcutaneous injection was made 7 days after the mouse environmental adaptation period, and the mice were randomly divided into two groups (n = 7 per group; Con (Control, control cells), STC1 OE (STC1 overexpressing, STC1 overexpressing cells). Control group on the flank of the mice. and STC1 overexpressing cells (1×10 6 cells) were injected and body weight was measured every week. (b) After the last measurement, the mouse was sacrificed and tumor tissue was recovered. The results of tumor volume measurement using calipers for 35 days are shown. The picture on the right is an image of a tumor surgically removed from a mouse. (c) H&E and staining results confirming STC1 expression immunohistochemically in mouse tumor tissue by injection of STC1-overexpressing cells. *, P <0.05; ** , P <0.01; ***, P < 0.001.
Figure 8 shows that STC1 overexpression promotes tumor metastasis to the lung in vivo. (a) This is a process diagram showing the mouse tumor metastasis experiment process. Intravenous injection was done 7 days after the mouse environmental adaptation period, and mice were randomly divided into two groups (n = 7 per group; Con (Control, control cells), STC1 OE (STC1 overexpressing, STC1 overexpressing cells). Tail of mice. Control and STC1-overexpressing cells (5 × 10 5 cells) were injected intravenously, and body weight was measured every week. (b) This is the result of confirming the image and number of mouse lung tissues in which lung metastases formed by injection of control and STC1-overexpressing cells were confirmed. The picture on the right shows the results of H&E, Ki67, and STC1 confirmed using immunohistochemical staining. (c) H&E staining and the level of STC1 expression in bladder cancer tissues of grade 1, 2, and 3 bladder cancer patients using immunohistochemical staining. This is the result shown by *, P < 0.05.
Figure 9 shows the results confirming that migration and invasion of bladder cancer cells treated with the conditioned medium of STC1 overexpressing cells were promoted and secretory STC1 protein expression was confirmed in the bladder cancer cells. (a) The left side shows the results of confirming STC1 protein in the conditioned medium of control and STC1 overexpressing cells. The right side shows the results of the MTT assay of bladder cancer cells treated with conditioned medium from control and STC1 overexpressing cells. (b) This shows that the colony forming ability of bladder cancer cells treated with conditioned medium from control and STC1 overexpressing cells was improved. (c) Shows the results of invasion and migration assays of bladder cancer cells treated with conditioned media of control and STC1 overexpressing cells. (d) An image of the wound area (%) recovered 24 hours later after a wound was made on a plate cultured with bladder cancer cells and treated with conditioned medium of control and STC1 overexpressing cells. On the right are the results of Western blot analysis of EMT markers in bladder cancer cells treated with conditioned medium from control and STC1 overexpressing cells. (e) Results of ELISA assay for secretory STC1 protein expression in P0 and P15 cells, control and STC1 overexpression vector-transfected cells, and control and STC1 overexpression cells. (f) This is the result of confirming secreted STC1 protein in the mouse serum identified in Figure 10 through ELISA assay. **, P <0.01; ***, P < 0.001.
Figure 10 shows that secretory STC1 protein increases cell proliferation and metastatic ability in bladder cancer cells. (a) This is the result of confirming the proliferation of cells at P0 and P15 treated with recombinant human STC1 (rhSTC1) in medium through MTT assay. (b) This is the result of confirming colony formation, cell invasion, and migration ability in P0 cells by adding rhSTC1 to the medium. (c) This is the result of confirming the invasion and migration ability of bladder cancer cells increased by rhSTC1, neutralized by STC1 antibody (ab). The picture on the right is the Western blot result confirming p-FAK activation by rhSTC1. *, P <0.05; **, P < 0.01
Figure 11 shows the results confirming the possibility as a biomarker by confirming secretory STC1 protein in serum and urine samples of bladder cancer patients. (a) Shows the STC1 concentration confirmed through ELISA assay in serum samples from healthy individuals and bladder cancer patients. Scatter plots containing data are indicated to indicate the range and distribution of measured levels of STC1 in serum by ELISA assay. Shows the results of comparing bladder cancer patients divided by grade, stage, and recurrence. (b) Shows the concentration of STC1 confirmed through ELISA assay in urine samples from healthy individuals and bladder cancer patients. Scatter plots containing data are indicated to indicate the range and distribution of measured levels of STC1 in urine by ELISA assay. Shows the results of comparing bladder cancer patients divided by grade, stage, and recurrence. ***, P < 0.001.
이하 첨부된 도면을 참조하여 본 발명의 실시예들을 상세히 설명한다. 이하의 설명에 있어, 당업자에게 주지 저명한 기술에 대해서는 그 상세한 설명을 생략할 수 있다. 또한, 본 발명을 설명함에 있어서, 관련된 공지 기능 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있다. 또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다.Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of techniques well known to those skilled in the art may be omitted. Additionally, when describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs.
따라서 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.
본 발명은 혈청 또는 소변에서 방광암 진단, 전이 또는 예후 예측을 위한STC1(Stanniocalcin-1) 바이오마커를 제공한다.The present invention provides a STC1 (Stanniocalcin-1) biomarker for diagnosing, metastasizing, or predicting prognosis of bladder cancer in serum or urine.
본 발명의 “STC1(Stanniocalcin-1)”은 뼈 물고기(bony fishe)에서 처음 발견된 호르몬인 stanniocalcin의 상동체인 당 단백질로서, 인간에서는 STC1 유전자에 의해 코딩되며, 매우 다양한 조직에서 발현되고 자가분비 또는 파라크린 기능을 가질 수 있는 분비된 동종이량체 당단백질 암호화한다. 현재까지 인간 STC1의 알려진 기능은 SUMOylation 경로에서 SUMO E3 유비퀴틴 리가아제 활성만이 보고되었다. STC1은 세포질, 미토콘드리아, 소포체 및 세포핵에서 많은 단백질과 상호작용하는 것으로 알려져 있다.“STC1 (Stanniocalcin-1)” of the present invention is a glycoprotein that is a homologue of stanniocalcin, a hormone first discovered in bony fish. In humans, it is encoded by the STC1 gene, and is expressed in a wide variety of tissues and is secreted through autocrine or It encodes a secreted homodimeric glycoprotein that may have paracrine functions. To date, the only known function of human STC1 has been reported as SUMO E3 ubiquitin ligase activity in the SUMOylation pathway. STC1 is known to interact with many proteins in the cytoplasm, mitochondria, endoplasmic reticulum, and cell nucleus.
본 발명에서, 사용된 용어 “진단”은 특정 질병 또는 질환에 대한 한 객체의 감수성(susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는 지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 객체의 예후(prognosis)(예컨대, 전-전이성 또는 전이성 암 상태의 동정, 암의 단계 결정 또는 치료에 대한 암의 반응성 결정)를 판정하는 것, 또는 테라메트릭스(therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링 하는 것)을 포함한다.In the present invention, the term “diagnosis” used refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or condition, a specific disease or Determining the prognosis of a diseased subject (e.g., identifying a pre-metastatic or metastatic cancer state, determining the stage of the cancer, or determining the responsiveness of the cancer to treatment), or therametrics (e.g., and monitoring the condition of the subject to provide information about treatment efficacy.
용어, "예후"는 암이 진단된 이후에 암의 완치 가능성, 치료 후 재발 가능성, 환자의 생존 가능성 등 암에 따른 환자의 각종 상태를 예측하는 것을 말한다. 암의 예후는 다양한 관점에서 추정될 수 있지만, 대표적으로 재발가능성, 생존가능성, 무병생존가능성의 관점에서 판단될 수 있다. 본 발명의 목적상 예후는 암의 진단 이후 생존의 예후를 의미할 수 있다. 본 발명에서 제공하는 바이오 마커를 사용하면, 암 환자의 생존 예후를 보다 용이하게 예측할 수 있어, 고 위험군의 환자를 분류하거나, 추가 필요한 치료 방법의 사용 여부를 결정하는 데 활용할 수 있고, 이로써 암 발병 후의 생존율을 높이는 데 기여할 수 있다.The term “prognosis” refers to predicting various conditions of a patient due to cancer after the cancer is diagnosed, such as the possibility of curing the cancer, the possibility of recurrence after treatment, and the patient's possibility of survival. The prognosis of cancer can be estimated from various perspectives, but can be typically judged from the perspectives of recurrence probability, survivability, and disease-free survival. For the purposes of the present invention, prognosis may mean the prognosis of survival after diagnosis of cancer. Using the biomarker provided by the present invention, the survival prognosis of cancer patients can be more easily predicted, which can be used to classify patients into high-risk groups or determine whether to use additional treatment methods, thereby increasing the incidence of cancer. It can contribute to increasing the survival rate later in life.
본 발명에서 용어 "(바이오)마커, 진단하기 위한 마커 또는 진단 마커(diagnosis marker)"란 암이 발생한 세포 또는 조직을 정상 세포 또는 조직과 구분하여 판정할 수 있는 물질로, 정상 세포에 비하여 암을 가진 세포에서 증가 양상을 보이는 폴리펩타이드 또는 핵산(예: mRNA 등), 지질, 당지질, 당단백질, 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자 등을 포함한다.In the present invention, the term "(bio)marker, marker for diagnosis, or diagnosis marker" refers to a substance that can be used to distinguish cancerous cells or tissues from normal cells or tissues. It includes organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, and sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase in cells with phosphorylation.
또한, 본 발명은 STC1(Stanniocalcin-1)의 발현수준을 측정할 수 있는 제제를 포함하는, 방광암(bladder cancer)의 진단, 전이 또는 예후 예측용 바이오마커 조성물을 제공한다.Additionally, the present invention provides a biomarker composition for diagnosing, metastasizing, or predicting prognosis of bladder cancer, including an agent capable of measuring the expression level of STC1 (Stanniocalcin-1).
상기 조성물은 STC1의 유전자 또는 단백질 발현수준을 측정하는 것이며, 상기 유전자 또는 이의 단편의 발현 수준을 확인하는 방법에 사용되는 제제는 시료에 포함된 해당 miRNA 또는 이의 단편의 발현 여부를 확인하는 방법에 사용되는 제제를 의미하는데, 예를 들어, RT-PCR, 경쟁적 RTPCR(Competitive RT-PCR), 실시간 RT-PCR(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블럿팅(Northern blotting), 유전자 칩 분석법 등의 방법에 사용되는 표적 유전자에 특이적으로 결합할 수 있는 프라이머, 프로브 또는 항체가 될 수 있으나, 특별히 이에 제한되지는 않는다.The composition measures the expression level of the gene or protein of STC1, and the agent used in the method for confirming the expression level of the gene or fragment thereof is used in the method for confirming the expression of the corresponding miRNA or fragment thereof contained in the sample. This refers to agents that are used, for example, RT-PCR, competitive RT-PCR (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting. It can be a primer, probe, or antibody that can specifically bind to a target gene used in methods such as Northern blotting and gene chip analysis, but is not particularly limited thereto.
본 발명에서 사용되는 용어 "프라이머"란, 짧은 자유 3말단 수산화기(free 3' hydroxylgroup)를 가지는 핵산서열로 상보적인 템플레이트(template)와 염기쌍(base pair)을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응(즉, DNA 폴리머레이즈 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성을 개시할 수 있다.The term "primer" used in the present invention refers to a nucleic acid sequence having a short free 3' hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template strand. It refers to a short nucleic acid sequence that functions as a point. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer and temperature.
본 발명에서 사용되는 용어 "프로브"란, 유전자 또는 mRNA와 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하는데, 올리고뉴클레오티드(oligonucleotide) 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있고, 보다 용이하게 검출하기 위하여 라벨링될 수 있다.The term "probe" as used in the present invention refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundreds of bases and can form a specific binding to a gene or mRNA, and is called an oligonucleotide. It can be manufactured in the form of a probe, single stranded DNA probe, double stranded DNA probe, RNA probe, etc., and can be labeled for easier detection.
상기 단백질의 발현 수준을 측정할 수 있는 제제는 상기 STC1에 특이적으로 결합하는 항체, 엡타머, 올리고펩타이드 또는 PNA(Peptide nucleic acid), 또는 상기 단백질을 코딩하는 유전자에 특이적인 상보적 서열을 갖는 프라이머 또는 프로브 등을 포함할 수 있으나, 이에 제한되지 않는다.Agents capable of measuring the expression level of the protein include antibodies, aptamers, oligopeptides, or PNA (Peptide nucleic acid) that specifically bind to the STC1, or have a complementary sequence specific to the gene encoding the protein. It may include primers or probes, but is not limited thereto.
본 발명의 일실시예에 따르면, 상기 STC1은 서열번호 1의 염기서열을 포함하는 것일 수 있다.According to one embodiment of the present invention, the STC1 may include the base sequence of SEQ ID NO: 1.
본 명세서에 사용되는 "폴리뉴클레오타이드" (또는 뉴클레오타이드, 핵산)는 DNA(gDNA 및 cDNA) 그리고 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 핵산 분자에서 기본 구성 단위인 뉴클레오타이드는 자연의 뉴클레오타이드 뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogues)도 포함한다.As used herein, “polynucleotide” (or nucleotide, nucleic acid) is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also sugars. Alternatively, it also includes analogues with modified base sites.
본 발명의 폴리뉴클레오타이드는 특정의 아미노산 서열(폴리펩타이드)을 암호화하는 핵산 분자에 제한되지않고, 특정 아미노산 서열에 대하여 실질적인 동일성을 나타내는 아미노산 서열 또는 그에 상응하는 기능을 갖는 폴리펩타이드를 암호화하는 핵산 분자를 포함하는 것으로 해석된다.The polynucleotide of the present invention is not limited to nucleic acid molecules encoding a specific amino acid sequence (polypeptide), but includes a nucleic acid molecule encoding an amino acid sequence showing substantial identity to a specific amino acid sequence or a polypeptide having a function corresponding thereto. It is interpreted as including.
본 발명의 일실시예에 따르면, 상기 STC1의 발현이 대조군의 기준치와 비교하여 증가되면, 암세포의 성장, 침습(invasion) 또는 이동(migration)이 증가되는 것일 수 있다.According to one embodiment of the present invention, when the expression of STC1 is increased compared to the baseline value of the control group, the growth, invasion, or migration of cancer cells may be increased.
본 발명의 일실시예에 따르면, 상기 STC1의 발현이 대조군의 기준치와 비교하여 증가되면, 암의 임상 병기(clinical stage)가 증가되는 것일 수 있다.According to one embodiment of the present invention, when the expression of STC1 is increased compared to the baseline value of the control group, the clinical stage of cancer may be increased.
본 발명에 따른 암 진단 및 예후 추정 방법은 암의 심각성 정도(임상 병기)를 판단하는데 사용될 수 있다. 예를 들면, 양성 대조군 및 음성 대조군의 프로파일과 비교하여, 경증, 중간 정도 또는 중증으로 평가될 수 있다. 나아가 일정한 암 집단에 대한 마커 프로파일 분석을 수행하여, 프로파일 결과를 근거로 일정 기준에 따라 분류할 수 있다.The cancer diagnosis and prognosis estimation method according to the present invention can be used to determine the severity level (clinical stage) of cancer. For example, compared to the profiles of positive and negative controls, it can be assessed as mild, moderate or severe. Furthermore, marker profile analysis can be performed on a certain cancer group and classified according to certain criteria based on the profile results.
본 발명의 일실시예에 따르면, 상기 STC1은 개체로부터 분리된 시료에서 측정되는 것일 수 있으며, 상기 시료는 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 및 소변으로 이루어진 군에서 선택되는 것일 수 있으나, 바람직하게는 혈청 또는 소변이나 이에 제한되지는 않는다.According to one embodiment of the present invention, the STC1 may be measured in a sample isolated from an individual, and the sample is selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, and urine. It may be, but is preferably serum or urine, but is not limited thereto.
또한, 본 발명은 상기의 조성물을 포함하는 방광암(bladder cancer)의 진단, 전이 또는 예후 예측용 키트를 제공한다.Additionally, the present invention provides a kit for diagnosing, metastasizing, or predicting prognosis of bladder cancer, comprising the composition described above.
본 발명의 용어 "키트"란, 특정한 목적을 위해 필요한 조성물 및 부속품들을 모아놓은 세트를 의미한다. 본 발명의 목적상, 본 발명의 키트는 방광암의 진단 또는 예후를 확인하는 것이다. 본 발명의 키트에는 방광암의 진단 또는 예후를 확인하기 위한 프라이머, 프로브, 선택적으로 펩타이드를 인지하는 항체 또는 방광암 발병시 특이적으로 발현이 변화되는 특정 펩타이드를 인지하는 항체뿐만 아니라 분석방법에 적합한 한 종류 또는 그 이상의 다른 구성성분 조성물, 용액 또는 장치가 포함될 수 있다.The term "kit" as used herein refers to a set of compositions and accessories required for a specific purpose. For the purpose of the present invention, the kit of the present invention is to confirm the diagnosis or prognosis of bladder cancer. The kit of the present invention includes primers, probes, antibodies that selectively recognize peptides or antibodies that recognize specific peptides whose expression is specifically changed when bladder cancer develops, as well as one type suitable for the analysis method for confirming the diagnosis or prognosis of bladder cancer. Or more other component compositions, solutions, or devices may be included.
또한, 본 발명은 개체로부터 생물학적 시료를 분리하는 단계;Additionally, the present invention includes the steps of isolating a biological sample from an individual;
상기 분리된 생물학적 시료에서 STC1(Stanniocalcin-1)의 발현수준을 측정하는 단계; 및Measuring the expression level of STC1 (Stanniocalcin-1) in the separated biological sample; and
상기 STC1의 발현 수준을 대조군의 기준치와 비교하는 단계;를 포함하는 방광암(bladder cancer)의 진단, 전이 또는 예후 예측을 위한 정보 제공 방법을 제공한다.It provides a method of providing information for diagnosis, metastasis, or prognosis prediction of bladder cancer, including the step of comparing the expression level of STC1 with a reference value of a control group.
본 발명의 일실시예에 따르면, 상기 생물학적 시료는 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 및 소변으로 이루어진 군에서 선택되는 것일 수 있고, 바람직하게는 혈청 또는 소변이나, 이에 제한되지는 않는다.According to one embodiment of the present invention, the biological sample may be selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, and urine, and is preferably serum or urine, but is limited thereto. It doesn't work.
본 발명의 일실시예에 따르면, 상기 STC1의 발현이 대조군의 기준치와 비교하여 증가되면, 방광암인 것으로 판단하는 것일 수 있다.According to one embodiment of the present invention, if the expression of STC1 increases compared to the baseline value of the control group, it may be determined that the cancer is bladder cancer.
본 발명의 일실시예에 따르면, 상기 STC1의 발현이 대조군의 기준치와 비교하여 증가되면, 암세포의 성장, 침습(invasion) 또는 이동(migration)이 증가된 것으로 판단하는 것일 수 있다.According to one embodiment of the present invention, if the expression of STC1 is increased compared to the baseline value of the control group, it may be determined that the growth, invasion, or migration of cancer cells is increased.
본 발명의 일실시예에 따르면, 상기 STC1의 발현이 대조군의 기준치와 비교하여 증가되면, 암의 임상 병기(clinical stage)가 증가된 것으로 판단하는 것일 수 있다.According to one embodiment of the present invention, if the expression of STC1 increases compared to the baseline value of the control group, it may be determined that the clinical stage of cancer has increased.
또한, 본 발명은 개체로부터 생물학적 시료를 분리하는 단계;Additionally, the present invention includes the steps of isolating a biological sample from an individual;
상기 분리된 생물학적 시료에 후보 물질을 처리하는 단계;Processing the separated biological sample with a candidate substance;
상기 후보물질이 처리된 생물학적 시료에서 STC1(Stanniocalcin-1)의 발현 수준을 측정하는 단계; 및Measuring the expression level of STC1 (Stanniocalcin-1) in the biological sample treated with the candidate material; and
상기 STC1의 발현 수준을 대조군의 기준치와 비교하는 단계;를 포함하는 방광암(bladder cancer) 치료제의 스크리닝 방법을 제공한다.It provides a screening method for a treatment for bladder cancer, comprising comparing the expression level of STC1 with a reference value of a control group.
본 발명의 일실시예에 따르면, 상기 STC1의 발현이 대조군의 기준치와 비교하여 저발현 되면, 항암효과가 있는 것으로 판단하는 것일 수 있다.According to one embodiment of the present invention, if the expression of STC1 is low compared to the reference value of the control group, it may be determined that there is an anticancer effect.
이하 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 하기 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail through examples. The following examples are merely for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
실시예 1. 혈청 또는 소변에서 검출가능한 방광암 바이오마커 선별을 위한 준비Example 1. Preparation for screening bladder cancer biomarkers detectable in serum or urine
1. 세포 배양1. Cell culture
인간 방광암 세포주인 T24, 5637, UC3, UC5 및 UC14는 American Type Culture Collection (ATCC)에서 구입하고 RT4 세포주는 Korean Cell Line Bank (KCLB)에서 구입하였다. T24, UC3, UC5, UC14 및 RT4 세포주는 DMEM(Dulbecco’s modified Eagle’s medium)에 배양하고 5637 세포주는 10% FBS (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) 및 1% penicillin/streptomycin (Capricorn Scientific GmbH, Ebsdorfergrund, Germany)이 첨가된 RPMI 1640에 배양하였다. 모든 세포주는 37℃, 5% CO2의 습한 대기에서 배양하였다. Human bladder cancer cell lines T24, 5637, UC3, UC5, and UC14 were purchased from American Type Culture Collection (ATCC), and RT4 cell line was purchased from Korean Cell Line Bank (KCLB). T24, UC3, UC5, UC14 and RT4 cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) and 5637 cell lines were cultured in 10% FBS (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and 1% penicillin/streptomycin (Capricorn Scientific GmbH, Ebsdorfergrund, Germany). ) was cultured in RPMI 1640 added. All cell lines were cultured at 37°C in a humidified atmosphere of 5% CO 2 .
2. 조정 배지(Conditioned Medium; CM)로부터 분비된 단백질의 수확2. Harvest of secreted proteins from Conditioned Medium (CM)
무혈청 조정 배지(CM)를 6시간 동안 40 ml의 무혈청 배지로 배양된 T24(P0), P15 (150 mm 디쉬)로 제조하였다. 배지를 수집하고 1,000 rpm에서 10분간 세포 잔해를 제거하였다. 조정 배지를 4℃에서 2시간 동안 3,850 rpm에서 VIVASPIN (GE Healthcare, USA)으로 농축하였다.Serum-free conditioned medium (CM) was prepared with T24 (P0) and P15 (150 mm dishes) cultured in 40 ml of serum-free medium for 6 hours. The medium was collected and cell debris was removed at 1,000 rpm for 10 minutes. The conditioned medium was concentrated with VIVASPIN (GE Healthcare, USA) at 3,850 rpm for 2 hours at 4°C.
3. LC-MS/MS에 의한 프로테오믹 분석3. Proteomic analysis by LC-MS/MS
단백질 농도를 BCA 검정으로 확인하고 추후 연구를 위해 샘플을 -70℃에서 보관하였다. 10 ㎍의 단백질 샘플을 12% SDS-PAGE 겔로 분리하고, 이 겔을 Coomassie Brilliant Blue R-250 buffer로 염색하였다. 이전 문헌에 기재된 방법에 따라 인-겔 분해(In-gel digestion)를 구성하였다 [Schevchenko A. et al., Nature Protocols 2006;1(6)2856-2860]. 겔을 분자량에 따라 4 부분으로 나누었다. 겔 분획을 탈염한 후 단백질의 시스테인의 환원 및 알킬화 후 트립신으로 분해하였다. 분해된 펩티드를 추출 용액 버퍼로 추출하였다. 분해된 펩티드를 0.02% 포름산 및 0.5% 아세트산을 함유하는 10 ㎕의 샘플 용액에 용해시켰다. LC-MS/MS 분석을 각 샘플마다 적어도 3회 실시하였다. Protein concentration was confirmed by BCA assay and samples were stored at -70°C for further studies. 10 μg of protein sample was separated on a 12% SDS-PAGE gel, and this gel was stained with Coomassie Brilliant Blue R-250 buffer. In-gel digestion was constructed according to the method described in the previous literature [Schevchenko A. et al., Nature Protocols 2006;1(6)2856-2860]. The gel was divided into four parts according to molecular weight. After desalting the gel fraction, the cysteine of the protein was reduced and alkylated, and then digested with trypsin. The degraded peptide was extracted with extraction solution buffer. The digested peptides were dissolved in 10 μl of sample solution containing 0.02% formic acid and 0.5% acetic acid. LC-MS/MS analysis was performed at least three times for each sample.
4. STC1 과발현 벡터 구성 및 Small-interference RNAs (siRNA)를 이용한 녹다운4. Construction of STC1 overexpression vector and knockdown using small-interference RNAs (siRNA)
인간 STC1을 암호화하는 플라스미드 발현 벡터의 트랜스펙션을 위해, STC1의 서열을 코딩하는 cDNA를 기질로서 정상 인간 조직으로부터 RT-PCR로 클로닝하고 PCR 산물을 pcDNA/His B 벡터로 서브클로닝하였다. HindIII-BamHI 제한 부위 측면의 STC1 오픈 리딩 프레임을 포함하는 DNA 시퀀싱을 T24 세포로부터 PCR 증폭하였다. 내인성 STC1의 녹다운을 위해, 세포를 siSTC1 올리고뉴클레오티드로 트렌스펙션시켰다. siSTC1 올리고뉴클레오티드를 Dharmacon SMARTPool에서 구입하였다. scRNA(scrambled siRNA) 또는 siSTC1 트랜스펙션을 ~100 nM의 최종 siRNA 농도에서 실시하였다. 녹다운 효율을 각각 qRT-PCR 또는 웨스턴 블롯 분석을 이용해 확인하였다.For transfection of the plasmid expression vector encoding human STC1, cDNA encoding the sequence of STC1 was cloned by RT-PCR from normal human tissue as a substrate, and the PCR product was subcloned into the pcDNA/His B vector. DNA sequencing containing the STC1 open reading frame flanked by Hin dIII- Bam HI restriction sites was PCR amplified from T24 cells. For knockdown of endogenous STC1, cells were transfected with siSTC1 oligonucleotide. siSTC1 oligonucleotide was purchased from Dharmacon SMARTPool. Scrambled siRNA (scRNA) or siSTC1 transfections were performed at a final siRNA concentration of ~100 nM. Knockdown efficiency was confirmed using qRT-PCR or Western blot analysis, respectively.
5. MTT 및 콜로니 형성 검정5. MTT and colony formation assay
1 x 103 세포를 96-웰 플레이트의 각 웰에 배양하였다. DMEM를 제거하고 세포 시간 위치를 기록하고 이동 비율을 0, 24, 48 및 72 시간 후에 각각 측정하였다. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)를 사용하여 각 웰에 첨가한 후 1시간 동안 인큐베이션하고 Dimethyl Sulfoxide (DMSO)를 첨가하였다. 540 nm에서의 흡광도를 spectrophotometer microplate reader로 측정하고 세포 생존률을 대조 세포와 비교한 퍼센트로 계산하였다.1 x 10 3 cells were cultured in each well of a 96-well plate. DMEM was removed, cell temporal positions were recorded, and migration rates were measured after 0, 24, 48, and 72 h, respectively. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well, incubated for 1 hour, and Dimethyl Sulfoxide (DMSO) was added. Absorbance at 540 nm was measured using a spectrophotometer microplate reader, and cell viability was calculated as a percentage compared to control cells.
1 x 103 세포를 6-웰 플레이트의 각 웰에 배양하고 눈에 보이는 콜로니가 형성될 때까지 7일까지 배양하였다. 콜로니를 4% 포름알데히드로 10분간 고정하고 0.1% 크리스탈 바이올렛 용액을 1시간 동안 각각 염색하였다. 콜로니 수를 Image J software를 사용하여 수동으로 계수하였다.1 x 10 3 cells were cultured in each well of a 6-well plate and cultured for up to 7 days until visible colonies were formed. Colonies were fixed with 4% formaldehyde for 10 minutes and stained with 0.1% crystal violet solution for 1 hour. Colony numbers were counted manually using Image J software.
6. 세포 침입 및 이동 검정6. Cell invasion and migration assay
세포의 침입 능력을 트렌스웰 검정을 사용하여 Boyden chamber에서 측정하였다. 4×104 세포를 매트리겔(matrigel) 코팅된 챔버에 로딩한 후 24시간 동안 배양하였다. 세포의 조정배지(CM)에 의한 세포 침입 분석의 경우에, CM에 의한 세포의 침입 또는 이동 능력을 확인하기 위해, 세포를 1:1 비율의 기본 조성 및 조정배지에서 24시간 동안 처리하여 침입 또는 이동 능력을 확인하였다.The invasion ability of cells was measured in a Boyden chamber using a transwell assay. 4×10 4 cells were loaded into a matrigel-coated chamber and cultured for 24 hours. In the case of cell invasion analysis by conditioned medium (CM), in order to confirm the invasion or migration ability of cells by CM, cells were treated in basic composition and conditioned medium at a 1:1 ratio for 24 hours to invade or migrate. Movement ability was confirmed.
7. 상처 회복 분석(Wound Healing Assay)7. Wound Healing Assay
세포를 6-웰 플레이트에 접종하고 90% 까지 가득 찰 때까지 24시간 동안 배양하였다. P200 피펫의 옐로우 팁으로 플레이트의 표면에 상처를 만든 후, 세포를 PBS로 여러 번 세척하여 세포 잔해를 제거하고 세포를 5% CO2, 37℃에서 배양하였다. 24시간 후, 세포를 광현미경으로 시각화시켰다. 그 후, 상처난 부분의 사진을 시간 간격을 두고 촬영하였다. 3개의 무작위 필드를 표시하여 측정하였다. 이동 인덱스를 대조 세포에 대한 처리 세포의 이동 거리의 비율로 표시하였다.Cells were seeded in 6-well plates and cultured for 24 hours until 90% confluent. After creating a wound on the surface of the plate with the yellow tip of a P200 pipette, the cells were washed several times with PBS to remove cell debris, and the cells were cultured at 37°C in 5% CO 2 . After 24 hours, cells were visualized by light microscopy. Afterwards, pictures of the wounded area were taken at intervals. Three random fields were marked and measured. The migration index was expressed as the ratio of the migration distance of treated cells to that of control cells.
8. qRT-PCR(Quantitative real-time polymerase chain reaction)8. qRT-PCR (Quantitative real-time polymerase chain reaction)
총 RNA를 RNAiso reagent (Takara)를 사용하여 분리하였다. RNA 정량 체크를 spectrophotometer (ND-1000)를 사용하여 평가하였다. 일차 가닥 cDNA 합성을 PrimeScriptTM RT reagent Kit (Takara)를 사용하여 1 ㎍ 총 RNA로부터 평가하였다. qRT-PCR을 TB Green Premix Ex Taq (Takara) 및 CFX 96 real-time PCR Detection system (BioRad)을 이용하여 실시하였다. 사용된 프라이머 세트 서열을 표 1에 나타냈다. 정량 평가의 재연성은 3회의 독립적인 cDNA 합성 및 RNA의 각 제조로부터의 PCR 증폭으로 평가하였다. mRNA 분석을 위해, 데이터를 내인성 대조로서 GAPDH에 대해 표준화하고 폴드 체인지(fold change)를 상대적 정량(2-ΔΔCt)을 통해 계산하였다.Total RNA was isolated using RNAiso reagent (Takara). RNA quantitative check was evaluated using a spectrophotometer (ND-1000). First-strand cDNA synthesis was assessed from 1 μg total RNA using the PrimeScript ™ RT reagent Kit (Takara). qRT-PCR was performed using TB Green Premix Ex Taq (Takara) and CFX 96 real-time PCR Detection system (BioRad). The primer set sequences used are shown in Table 1. The reproducibility of the quantitative assessment was assessed by three independent cDNA syntheses and PCR amplification from each preparation of RNA. For mRNA analysis, data were normalized to GAPDH as an endogenous control and fold change was calculated via relative quantification (2 -ΔΔCt ).
9. 웨스턴 블롯 분석 및 항체 9. Western blot analysis and antibodies
웨스턴 블롯 분석을 제조사의 지시사항에 따라 실시하였다. 세포를 PBS를 사용해 첫 번째로 세척한 후 단백질을 radio immunoprecipitation (RIPA) buffer (Ambion, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, protease inhibitor cocktail, 및 phosphatase inhibitor)로 분리하고 원심분리하였다(12,000 g, 15 min, 4℃). 단백질의 양을 BCA assay kit (Thermo Fisher Scientific)를 사용하여 평가한 후, 10-12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE)를 실시하고 니트로셀룰로오스(NC) 맴브레인(GE healthcare)으로 전기적으로 옮겼다. 그런 다음 0.05% TBS-T 중의 5% 무지방 우유를 이용해 맴브레인을 차단하였다. STC1(Santa Cruz Biotech)에 대한 항체는 MMP-1 (Santa Cruz Biotech), MMP2 (Cell signaling), MMP9 (Cell signaling), NCAD (Cell signaling), ECAD (Cell signaling), VIM (Santa Cruz Biotech), SNAIL (Santa Cruz Biotech), FAK(Cell signaling), p-FAK (Cell signaling,), ERK (Cell signaling), 및 p-ERK (Cell signaling)이었다. GAPDH (Cell signaling)는 로딩 대조군으로서 사용하였다. horseradish peroxidase (HRP)-conjugated anti-rabbit 또는 anti-mouse immunoglobulin G (IgG)을 이차 항체로 사용하고 양성 밴드를 ECL 검출 시약을 사용하여 검출하였다. 화학형광 시그널의 최종 시각화를 automatic X-ray film processor (JPI Healthcare)로 캡쳐하고 X-ray films (Fuji film) 상의 시그널 강도를 Image J software로 정량하였다. Western blot analysis was performed according to the manufacturer's instructions. After the cells were first washed with PBS, the proteins were stored in radio immunoprecipitation (RIPA) buffer (Ambion, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, protease inhibitor cocktail, and phosphatase inhibitor). and centrifuged (12,000 g, 15 min, 4°C). After evaluating the amount of protein using the BCA assay kit (Thermo Fisher Scientific), 10-12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed and electrically transferred to a nitrocellulose (NC) membrane (GE healthcare). . The membrane was then blocked using 5% fat-free milk in 0.05% TBS-T. Antibodies against STC1 (Santa Cruz Biotech), MMP-1 (Santa Cruz Biotech), MMP2 (Cell signaling), MMP9 (Cell signaling), NCAD (Cell signaling), ECAD (Cell signaling), VIM (Santa Cruz Biotech), SNAIL (Santa Cruz Biotech), FAK (Cell signaling), p-FAK (Cell signaling), ERK (Cell signaling), and p-ERK (Cell signaling). GAPDH (Cell signaling) was used as a loading control. horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG) was used as a secondary antibody, and positive bands were detected using ECL detection reagent. The final visualization of the chemifluorescence signal was captured with an automatic X-ray film processor (JPI Healthcare), and the signal intensity on X-ray films (Fuji film) was quantified with Image J software.
10. 생체 내 종양 성장 및 전이 능력 검정 10. In vivo tumor growth and metastatic ability assay
생체 내 종양 형성 및 전이 능력 검정을 위해, P0 또는 P15 세포를 트립신화시키고 PBS에 현탁하였다. 그런 다음, 세포를 각 BALB/C 누드 마우스의 측면 및 꼬리 정맥에 피하 주입하였다. 종양 형성 능력을 확인하기 위하여 세포를 200 ㎕ 부피의 PBS 중의 세포 및 동량의 Matrigel과 섞어서 피하로 주입하였다. 마우스의 체중 및 종양이 측정 가능한 시기에 캘리퍼스를 이용하여 측정하고 종양 부피를 계산하였다: Tumor volume (mm3)= width2 (mm2) x length (mm). 종양 조직에서 RNA 및 단백질 추출 시 마우스 조직을 PBS로 2회 세척하였으며, 측정 35일차에 마우스를 해부하여 조직을 수득하였다. 종양 전이 능력을 확인하기 위하여 꼬리 정맥에 주입한 마우스는 해부하여 폐 결절 형성 개수를 확인하였다. For in vivo tumor formation and metastatic ability assays, P0 or P15 cells were trypsinized and suspended in PBS. Cells were then injected subcutaneously into the lateral and tail veins of each BALB/C nude mouse. To confirm tumor formation ability, cells were mixed with 200 μl of cells in PBS and an equal amount of Matrigel and injected subcutaneously. At the time when the mouse's body weight and tumor could be measured, they were measured using calipers and the tumor volume was calculated: Tumor volume (mm 3 )=width 2 (mm 2 ) x length (mm). When extracting RNA and protein from tumor tissue, the mouse tissue was washed twice with PBS, and on the 35th day of measurement, the mouse was dissected to obtain the tissue. To confirm tumor metastasis ability, mice injected into the tail vein were dissected to determine the number of lung nodules formed.
11. 조직 마이크로어레이(Tissue Microarray; TMA) 및 면역조직화학(Immunohistochemistry; IHC) 11. Tissue Microarray (TMA) and Immunohistochemistry (IHC)
TMA 블록을 마우스의 파라핀-블록으로부터 조직 지름 2 mm로 선택하였다. 슬라이드를 H&E(hematoxylin 및 eosin)로 염색하고 대표 종양 조직을 확인하기 위해 관찰하였다. IHC에서, 모든 조직 샘플을 완충 포르말린(Sigma-Aldrich, St. Louis, MO, USA)에 고정하고 파라핀에 함침했다. 파라핀-함침 조직을 자일렌 내에서 탈파라핀화시키고 알코올(100 %, 90%, 80%, 및 60%)에서 재수화시켰다. 항원 회수(끓는 물에서 10분)를 실시하고, 시트르산나트륨을 pH 7 회수 버퍼로서 사용하였다. 사용된 일차 STC1 및 Ki67 항체(Santa Cruz Biotech)는 rabbit monoclonal IgG (Abcam)이었다. Rabbit IgG를 음성 아이소타입 대조로서 사용하였다. TMA 슬라이드를 일차 항체와 함께 4℃에서 처리하고 비오틴화된 이차 항체를 처리하였다. Vectastain Elite ABC Reagent (Vector Laboratories)를 실온에서 30분간 첨가하고 3, 3’-diaminobenzidine (DAB)을 색소원으로 사용하여 면역반응을 검출하였다. 그런 다음, TMA 슬라이드를 Mayer’s hematoxylin (Dako)로 대조염색하고, 알코올(60%, 80%, 90%, 및 100%)로 탈수하고, 자일렌으로 3회 세척하고 자일렌 중의 봉입제로 고정하였다. 현미경으로 염색 결과를 확인하였다.TMA blocks were selected from paraffin-blocks of mice with a tissue diameter of 2 mm. Slides were stained with H&E (hematoxylin and eosin) and observed to identify representative tumor tissues. For IHC, all tissue samples were fixed in buffered formalin (Sigma-Aldrich, St. Louis, MO, USA) and embedded in paraffin. Paraffin-impregnated tissues were deparaffinized in xylene and rehydrated in alcohol (100%, 90%, 80%, and 60%). Antigen retrieval (10 minutes in boiling water) was performed and sodium citrate was used as pH 7 retrieval buffer. The primary STC1 and Ki67 antibodies (Santa Cruz Biotech) used were rabbit monoclonal IgG (Abcam). Rabbit IgG was used as a negative isotype control. TMA slides were treated at 4°C with primary antibodies and biotinylated secondary antibodies. Vectastain Elite ABC Reagent (Vector Laboratories) was added at room temperature for 30 minutes, and the immune response was detected using 3, 3'-diaminobenzidine (DAB) as a chromogen. TMA slides were then counterstained with Mayer’s hematoxylin (Dako), dehydrated with alcohol (60%, 80%, 90%, and 100%), washed three times with xylene, and fixed with embedding agent in xylene. The staining results were confirmed under a microscope.
12. 인간 혈청 및 소변 샘플12. Human serum and urine samples
BC 환자의 혈액을 헤파린 첨가 식염수 튜브에 수집하고 3,000 rpm에서 10분간 원심분리하였다. 혈액으로부터 분리된 혈청을 냉동하여 보관하였다. 소변 샘플을 건강한 사람 및 방광 환자로부터 각각 수집하였다. 튜브 중에서 20 ㎖의 소변을 4℃에서 3,000 rpm으로 10분간 원심분리하였다. 소변의 상층액을 VIVASPIN 컬럼을 사용하여 농축하고 실험에 사용하였다.Blood from BC patients was collected in heparinized saline tubes and centrifuged at 3,000 rpm for 10 minutes. Serum separated from blood was frozen and stored. Urine samples were collected from healthy subjects and bladder patients respectively. 20 ml of urine in the tube was centrifuged at 3,000 rpm for 10 minutes at 4°C. The supernatant of urine was concentrated using a VIVASPIN column and used in the experiment.
13. 인간 STC1 ELISA 검정 13. Human STC1 ELISA assay
조정배지, 혈청 및 소변 샘플 중의 STC1의 농도를 Enzyme-linked immunosorbent assay (ELISA) kit (R&D systems)를 사용해 분석하였다. The concentration of STC1 in conditioned media, serum, and urine samples was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (R&D systems).
14. 환자 및 유전자 발현 데이터14. Patient and gene expression data
National Center for Biotechnology information (NCBI) Gene Expression Omnibus (GEO) 데이터베이스 (GSE13507, GSE32894, 및 GSE120736)로부터 임상 및 유전자 발현 데이터를 포함하는 데이터 세트를 수득하였다. 모든 데이터는 log2 스케일로 변환시키고 quantile normalization으로 표준화하였다. 165명의 방광암 환자 데이터를 디스커버리 코호트(n=165; Korean cohort; GSE13507)로 사용하고, 453명의 방광암 환자 데이터를 발리데이션 코호트(n=308; Lund cohort; GSE32894, n=145; Yonsei cohort; GSE120736)로 사용하였다.Data sets containing clinical and gene expression data were obtained from the National Center for Biotechnology information (NCBI) Gene Expression Omnibus (GEO) database (GSE13507, GSE32894, and GSE120736). All data were transformed to log2 scale and normalized by quantile normalization. Data from 165 bladder cancer patients were used as the discovery cohort (n=165; Korean cohort; GSE13507), and data from 453 bladder cancer patients were used as the validation cohort (n=308; Lund cohort; GSE32894, n=145; Yonsei cohort; GSE120736). It was used as.
15. 연관성, 유전자 발현 및 Function Enrichment 분석 15. Association, gene expression and function enrichment analysis
유전자 특성과 관련된 유의한 유전자 세트를 제조하기 위해, 한국 방광암 환자 코호트 (GSE13507)로부터의 유전자 발현 데이터에 Pearson correlation test를 적용하고 유의한 연관성 계수(|r| > 0.4 및 p < 0.001)를 갖는 유전자를 선택하였다. 유사성 및 완전 연결 클러스터링 방법(complete linkage clustering method)의 척도로서 중심 상관 계수로 계층 클러스터링 분석을 실시하였다. 환자 클러스터링 결과에 따라, 환자를 2개의 서브그룹으로 나누고, 각 서브그룹에서 환자의 진행 시간 및 암 특이 생존률을 평가하였다. Kaplan-Meier 방법을 사용하여 log-rank statistics로 무진행 시간 및 암 특이 생존을 계산하였다. Gene ontology (GO) 분석을 DAVID bioinformatic resources (http://david.ncifcrf.gov)로 실시하고, 결과는 p < 0.001 및 false discovery rate (FDR) < 0.25일 때 유의한 것으로 간주하였다.To prepare a set of significant genes associated with genetic characteristics, we applied the Pearson correlation test to gene expression data from the Korean bladder cancer patient cohort (GSE13507) and identified genes with significant correlation coefficients (|r| > 0.4 and p < 0.001). was selected. Hierarchical clustering analysis was performed with central correlation coefficient as a measure of similarity and complete linkage clustering method. According to the patient clustering results, patients were divided into two subgroups, and the progression time and cancer-specific survival rate of patients in each subgroup were evaluated. Progression-free time and cancer-specific survival were calculated using log-rank statistics using the Kaplan-Meier method. Gene ontology (GO) analysis was performed with DAVID bioinformatic resources (http://david.ncifcrf.gov), and results were considered significant when p < 0.001 and false discovery rate (FDR) < 0.25.
16. 통계 분석 16. Statistical analysis
데이터 결과는 3회 반복 연구의 평균 ± 표준 편차 (SD)로 나타냈다. 모든 분석은 적어도 3회 실시하였으며 3회의 개별 실험으로부터의 데이터로 나타냈다. 모든 실험은 3중 디바이스로 구성하였다. 모든 수치 데이터는 평균 ± S.D로 나타냈다. 두 독립 그룹 사이의 차이의 유의성은 two-tailed Student’s t-test를 사용하여 결정하였다. 차이는 P < 0.05에서 통계학적으로 유의한 것으로 간주하였다. * ,P < 0.05; **, P < 0.01; ***, P < 0.001. 통계 분석은 R 3.6.1 language environment (http://www.r-project.org)을 사용하여 실시하였다.Data results are expressed as mean ± standard deviation (SD) of three replicate studies. All analyzes were performed at least in triplicate and are presented as data from three separate experiments. All experiments consisted of a triple device. All numerical data are expressed as mean ± SD. The significance of differences between two independent groups was determined using the two-tailed Student's t -test. Differences were considered statistically significant at P < 0.05. * ,P <0.05; **, P <0.01; ***, P < 0.001. Statistical analysis was performed using R 3.6.1 language environment (http://www.r-project.org).
실시예 2. 혈청 또는 소변에서 검출 가능한 방광암 바이오마커의 검정Example 2. Assay for bladder cancer biomarkers detectable in serum or urine
1. 방광암 항암제 내성 세포의 조정 배지(CM)에서 분비된 단백질 분석1. Analysis of proteins secreted from conditioned medium (CM) of bladder cancer anticancer drug-resistant cells
방광암 항암제 내성 세포주에서 분비되는 단백질들을 확인하기 위해, P0 및 P15 세포의 조정배지로부터 샘플을 제조한 후 liquid chromatograph-tandem mass spectrophotometer(LC-MS/MS)를 실시하였다(도 1a). P0 및 P15 세포의 조정 배지에서 발현된 다량의 단백질들을 확인하였으며, 각각 662 및 805개의 분비성 단백질로 확인되었다(도 1b). P0와 비교하여 P15 세포의 조정배지에서만 분비된 단백질을 비교하고, 관련 경로를 확인하였다. 그 결과, ingenuity pathway analysis (IPA)에 의해 분석하였으며, 세포 이동과 관련된 단백질이 발현이 13개 기준 경로에서 현저하게 변화함을 발견하였다(도 1b). P0 및 P15 세포의 조정 배지 사이에서 차별적으로 발현하는 386개 단백질 중 P15 세포에서 과발현하면서 세포외 공간에 위치하는 총 27개 단백질을 확인하였다(도 1c).To identify proteins secreted from bladder cancer anticancer drug-resistant cell lines, samples were prepared from the conditioned medium of P0 and P15 cells and then liquid chromatograph-tandem mass spectrophotometer (LC-MS/MS) was performed (Figure 1a). A large number of proteins expressed in the conditioned medium of P0 and P15 cells were identified, with 662 and 805 secreted proteins, respectively (Figure 1b). Proteins secreted only in the conditioned medium of P15 cells compared to P0 were compared, and related pathways were identified. As a result, it was analyzed by ingenuity pathway analysis (IPA), and it was found that the expression of proteins related to cell migration changed significantly in 13 reference pathways (Figure 1b). Among the 386 proteins differentially expressed between the conditioned media of P0 and P15 cells, a total of 27 proteins were identified that were overexpressed in P15 cells and located in the extracellular space (Figure 1c).
2. 높은 STC1 발현과 방광암 환자들의 나쁜 예후 관련성 확인2. Confirmation of correlation between high STC1 expression and poor prognosis in bladder cancer patients
우선 STC1의 유전자 발현 수준을 확인하고 원발성 NMIBC, 원발성 MIBC을 포함하는 방광 조직, 및 방광암 코호트에서의 재발 조직에서의 발현 수준과 비교하였다. 다양한 방광암 코호트 (한국인 방광암 코호트, GSE13507, Lund 코호트, GSE32894, Yonsei 코호트, GSE120736)에서의 유전자 발현 데이터 비교에서, 모든 케이스에서 원발성 MIBC에서 STC1의 발현 수준이 원발성 NMIBC에 비해 상당히 높았다(P = 0.01, P < 0.001, 및 P = 0.05 by a two-sample t-test, 도 2a). 도 2b는 618명의 방광암 환자의 기본 특성을 나타낸 것이다. 한국인 방광암 코호트(GSE13507)에서, 평균 연령은 66세(24세 내지 88세 범위)이고, 수술 후 평균 후속 주기는 53개월(1개월 내지 161개월 범위)였다. 후속 주기 동안, 34명의 환자(21%)가 질환이 진행되었다. Lund 코호트(Lund cohort)에서, 평균 연령은 71세(20세 내지 96세 범위)이고, 수술 후 평균 후속 주기는 46개월(2개월 내지 127개월)이었다. 후속 주기 동안, 19명의 환자(12%)가 질환이 진행되었다. Yonsei 코호트(Yonsei cohort)에서, 평균 연령은 73세(36세 내지 100세 범위)이고, 수술 후 평균 후속 주기는 70개월(1개월 내지 103개월 범위)였다. 다만, Yonsei 코호트에서 무 진행 생존률 데이터는 공급받지 못했다. First, the gene expression level of STC1 was confirmed and compared with the expression level in primary NMIBC, bladder tissue including primary MIBC, and relapse tissue in the bladder cancer cohort. In comparison of gene expression data in various bladder cancer cohorts (Korean bladder cancer cohort, GSE13507; Lund cohort, GSE32894; Yonsei cohort, GSE120736), the expression level of STC1 in primary MIBC was significantly higher than that in primary NMIBC in all cases (P = 0.01; P < 0.001, and P = 0.05 by a two-sample t-test, Figure 2a). Figure 2b shows the baseline characteristics of 618 bladder cancer patients. In the Korean bladder cancer cohort (GSE13507), the mean age was 66 years (range 24 to 88 years) and the mean follow-up period after surgery was 53 months (range 1 to 161 months). During subsequent cycles, 34 patients (21%) had disease progression. In the Lund cohort, the mean age was 71 years (range 20 to 96 years) and the mean follow-up period after surgery was 46 months (range 2 to 127 months). During subsequent cycles, 19 patients (12%) had disease progression. In the Yonsei cohort, the mean age was 73 years (range 36 to 100 years) and the mean follow-up period after surgery was 70 months (range 1 to 103 months). However, progression-free survival data from the Yonsei cohort were not available.
STC1이 많은 암에서 흔히 상향조절되고 예후 마커로서 사용되므로, 방광암 환자의 생존 결과에서 STC1의 예상 수치를 추가 평가하였다. 이는 STC1 발현 수준과 직접적으로 관련된 유전자 발현 시그니처를 확인하고 질환 진행 및 생존의 가능성을 예측하기 위한 시그니처로 사용하기 위한 것이다. GSE13507 코호트에서 STC1 발현과 관련된 367개 유전자를 확인하였다(Pearson’s correlation test, P < 0.001, |r| > 0.4). 이러한 유전자의 발현 패턴의 계층 클러스터링 분석에 근거하여, 방광암 환자를 STC1-low 및 STC1-high의 2개 그룹으로 나누었다(도 2c). STC1-low 환자의 진행 생존률은 STC1-high 환자보다 상당히 높았다(log-rank test, P = 0.007; 도 2c). STC1-low 환자의 암-특이 생존률은 STC1-high 환자에 비해 상당히 높았다(log-rank test, P < 0.001; 도 2c). 본 발명에서는 또한 STC1 발현과 관련된 중요 시그널링 경로를 확인하였다. STC1 관련 367개 유전자의 GO 분석(279개 상향조절 유전자)을 DAVID software를 사용하여 실시하였다. 상향조절 유전자를 DAVID에 적용시켰을 때, 염증 반응, 세포외 매트릭스 조직화, 백혈구 이동, 세포 주화성, 시그널 트랜스덕션, 식균작용, 및 PI3K-Akt 시그널링 경로에 관련된 유전자가 상당히 풍부하였다(도 2d). 또한, STC1 발현에 밀접하게 관련된 21개 유전자도 확인하였다(도 2d). 독립적인 환자 코호트(GSE32894 및 GSE120736)로부터 유전자 발현 데이터를 사용하여 상기 발견을 입증하였다. Lund 코호트(GSE32984)에서의 시그니처-기반 계층 클러스터 분석과 동일한 과정을 통해, STC1-high 또는 STC1-low 그룹으로 방광암 환자를 계층화하였다(도 2e). STC1-low 환자의 진행 생존율은 STC1-high 환자보다 상당히 높았다(log-rank test, P = 0.001 및 P = 0.36; 도 2e). 또한, 방광암 환자를 Yonsei 코호트(GSE120736)에서의 두 그룹으로 계층화하였다. STC1-low 환자의 암-특이 생존율은 STC1-high 환자보다 상당히 더 높았다(log-rank test, P = 0.004; 도 2f).Because STC1 is commonly upregulated in many cancers and used as a prognostic marker, we further evaluated the expected level of STC1 in the survival outcomes of bladder cancer patients. This is to identify gene expression signatures directly related to STC1 expression levels and to use them as signatures to predict disease progression and likelihood of survival. In the GSE13507 cohort, 367 genes associated with STC1 expression were identified (Pearson’s correlation test, P < 0.001, |r| > 0.4). Based on hierarchical clustering analysis of the expression patterns of these genes, bladder cancer patients were divided into two groups: STC1-low and STC1-high (Figure 2c). The progression survival rate of STC1-low patients was significantly higher than that of STC1-high patients (log-rank test, P = 0.007; Fig. 2C). The cancer-specific survival rate of STC1-low patients was significantly higher than that of STC1-high patients (log-rank test, P < 0.001; Figure 2c). In the present invention, we also identified important signaling pathways related to STC1 expression. GO analysis of 367 STC1-related genes (279 upregulated genes) was performed using DAVID software. When the upregulated genes were subjected to DAVID, genes involved in inflammatory response, extracellular matrix organization, leukocyte migration, cell chemotaxis, signal transduction, phagocytosis, and PI3K-Akt signaling pathway were significantly enriched (Figure 2D). Additionally, 21 genes closely related to STC1 expression were identified (Figure 2d). The findings were validated using gene expression data from independent patient cohorts (GSE32894 and GSE120736). Bladder cancer patients were stratified into STC1-high or STC1-low groups through the same process as the signature-based hierarchical cluster analysis in the Lund cohort (GSE32984) (Figure 2e). The progression survival rate of STC1-low patients was significantly higher than that of STC1-high patients (log-rank test, P = 0.001 and P = 0.36; Figure 2e). Additionally, bladder cancer patients were stratified into two groups in the Yonsei cohort (GSE120736). The cancer-specific survival rate of STC1-low patients was significantly higher than that of STC1-high patients (log-rank test, P = 0.004; Figure 2f).
3. STC1의 방광암에서 세포의 증식, 이동 및 침습 능력을 촉진 확인3. Confirmation of STC1 promoting cell proliferation, migration, and invasion ability in bladder cancer
증가된 STC1 발현은 다양한 유형의 암 환자에서 불량한 예후와 관련되어 있다. STC1이 방광암에서 세포 증식에 기여하는지 여부를 확인하기 위해, P0 세포주에 STC1 과발현 벡터(pSTC1) 또는 STC1의 small-interference RNA(siSTC1)로 트랜스펙션 시킨 세포주로 우선 발현 증가 및 감소를 확인했다 (도 3a). STC1 과발현 및 녹다운 시킨 세포주를 이용하여 MTT(도 3b) 실험을 통한 세포 증식과 클론원성 검정(도 3c)을 통한 콜로니 형성 능력을 확인하였다. 두 결과에서 모두 STC1의 발현에 따라 세포의 증식을 조절하는 것을 확인하였다(도 3b, c). 또한 STC1 과발현 안정(STC1 overexpressing stable; STC1 OE) 세포주에서도 대조군 세포와 비교하여 STC1의 mRNA, 단백질 발현 및 세포의 증식, 콜로니 형성 능력, 침습 및 이동 능력모두 증가한 결과를 확인하였습니다(도 4a, b, c). 이러한 결과들을 통해 STC1가 방광암에서 세포 증식뿐만 아니라 이동 및 침습 능력도 촉진시킨다는 것을 제시한다. Increased STC1 expression is associated with poor prognosis in patients with various types of cancer. To determine whether STC1 contributes to cell proliferation in bladder cancer, the increase and decrease in expression was first confirmed in the P0 cell line transfected with the STC1 overexpression vector (pSTC1) or the small-interference RNA of STC1 (siSTC1) ( Figure 3a). Using cell lines overexpressing and knocking down STC1, cell proliferation through MTT (Figure 3b) experiment and colony formation ability through clonogenic assay (Figure 3c) were confirmed. Both results confirmed that cell proliferation was controlled by the expression of STC1 (Figures 3b, c). In addition, in the STC1 overexpressing stable (STC1 OE) cell line, the mRNA and protein expression of STC1, cell proliferation, colony formation ability, invasion and migration ability were all increased compared to control cells (Figures 4a, b, c). These results suggest that STC1 promotes not only cell proliferation but also migration and invasion ability in bladder cancer.
4. STC1의 epithelial-mesenchymal transition (EMT) 유전자들과의 상관관계 확인4. Confirmation of correlation of STC1 with epithelial-mesenchymal transition (EMT) genes
GSE13507 데이터에서 STC1와 연관된 EMT-관련 유전자인 VIM, ZEB1, ZEB2, SNAI1, TWIST1, TWIST2, MMP1, MMP3, MMP9, NCAD, 및 CD44는 STC1와 양의 상관관계를 보였으며 (도 5a), mesenchymal-epithelial transition(MET)-관련 유전자인 SDC1 및 ECAD은 STC1와 음의 상관관계를 보였다(도 5b). 다음으로는 STC1가 방광암 세포에서 이동 및 EMT 관련 유전자간의 발현 조절 여부를 확인하기 위해, MMP1, MMP2, MMP9, NCAD, VIM, SNAIL, SLUG, ZEB1, ZEB2 및 TWIST의 mRNA 발현이 증가됨을 확인하였다(도 6a). 웨스턴 블롯팅 결과를 통해, MMP1, MMP2, MMP9, NCAD, VIM, 및 SNAIL의 단백질 수준이 증가됨을 확인하였으며, 반대로 MET 관련 ECAD 단백질 수준이 감소됨을 확인하였다(도 6b).In the GSE13507 data, the EMT-related genes VIM, ZEB1, ZEB2, SNAI1, TWIST1, TWIST2, MMP1, MMP3, MMP9, NCAD, and CD44, which were associated with STC1, showed positive correlation with STC1 (Fig. 5a), and mesenchymal- The epithelial transition (MET)-related genes SDC1 and ECAD showed a negative correlation with STC1 (Figure 5b). Next, to determine whether STC1 regulates the expression of genes related to migration and EMT in bladder cancer cells, it was confirmed that the mRNA expression of MMP1, MMP2, MMP9, NCAD, VIM, SNAIL, SLUG, ZEB1, ZEB2, and TWIST was increased ( Figure 6a). Through Western blotting results, it was confirmed that the protein levels of MMP1, MMP2, MMP9, NCAD, VIM, and SNAIL were increased, and conversely, the MET-related ECAD protein level was confirmed to be decreased (Figure 6b).
5. STC1의 생체 내에서 방광암 종양 성장 및 폐 전이 증가 확인5. Confirmation of STC1 increased bladder cancer tumor growth and lung metastasis in vivo
대조군 세포 및 STC1 과발현 안정 세포를 수컷 BALB/C 누드 마우스의 옆구리 영역에 피하 주입하여 전체적인 실험 개략도는 다음과 같다(도 7a). STC1 과발현 안정 세포를 주입한 마우스 그룹의 체중은 대조군 마우스 그룹에 비해 유의하진 않지만 증가하였으며, 종양 크기는 급격히 증가하였다(도 7a, b). 도 9b는 대조군 및 STC1 과발현 안정 세포가 주입된 마우스의 옆구리에 형성된 종양의 대표 사진을 나타낸 것이다. STC1의 mRNA 발현은 대조군 세포 주입 마우스 그룹의 종양에서보다 STC1 과발현 안정 마우스 그룹에서 증가하였다(도 7c). 그러므로, IHC에 의해 확인된 STC1의 단백질 발현은 STC1 과발현 안정 세포주 주입 마우스 종양에서 증가하였다(도 7c). 이러한 결과들은 STC1가 생체 내에서 종양 성장을 증가시킴을 보인다. Control cells and STC1-overexpressing stable cells were injected subcutaneously into the flank area of male BALB/C nude mice. The overall experimental schematic is as follows (Figure 7a). The body weight of the mouse group injected with stable STC1-overexpressing cells increased, although not significantly, compared to the control mouse group, and the tumor size increased rapidly (Figure 7a, b). Figure 9b shows representative photographs of tumors formed on the flanks of mice injected with control and STC1-overexpressing stable cells. The mRNA expression of STC1 was increased in the STC1 overexpression stable mouse group than in the tumors of the control cell-injected mouse group (Figure 7c). Therefore, protein expression of STC1 confirmed by IHC was increased in mouse tumors injected with STC1 overexpressing stable cell line (Figure 7c). These results show that STC1 increases tumor growth in vivo.
그런 다음, STC1가 폐 전이를 조절하는지를 확인하기 위해, 대조군 및 STC1 과발현 안정 세포를 마우스 꼬리 정맥에 주입하여 전체적인 실험 개략도는 다음과 같다(도 8a). 두 그룹간의 체중 변화 및 폐 전이가 대조군의 마우스 그룹에 비해 STC1 과발현 안정 세포 마우스 그룹 모두 시간이 지남에 따라 증가됨을 관찰되었다(도 8a). STC1 과발현 안정 세포주가 주입된 마우스 그룹에서 대조군 세포 마우스 그룹에 비해 더 많고 큰 폐 결절이 확인되었다(도 8b). Ki67 및 STC1의 더 높은 발현이 STC1 과발현 안정 세포가 주입된 마우스의 폐 조직에서 확인되었다(도 8b). 또한, STC1 단백질 발현은 방광암 환자의 조직 샘플에서 Grade 1, 2, 3으로 나누어 확인하였으며, grade가 증가할수록 STC1의 발현 또한 증가하였다(도 8c). 이러한 결과들은 STC1은 종양 형성 및 폐 전이를 촉진시키는 중요한 역할을 시사한다.Then, to determine whether STC1 regulates lung metastasis, control and STC1-overexpressing stable cells were injected into the mouse tail vein. The overall experimental schematic is as follows (Figure 8a). It was observed that body weight change and lung metastasis between the two groups increased over time in both STC1-overexpressing stable cell mouse groups compared to the control mouse group (Figure 8a). More and larger lung nodules were identified in the mouse group injected with the STC1 overexpressing stable cell line compared to the control cell mouse group (Figure 8b). Higher expression of Ki67 and STC1 was confirmed in the lung tissue of mice injected with STC1-overexpressing stable cells (Figure 8B). In addition, STC1 protein expression was confirmed in tissue samples from bladder cancer patients divided into grades 1, 2, and 3, and as the grade increased, the expression of STC1 also increased (Figure 8c). These results suggest that STC1 plays an important role in promoting tumorigenesis and lung metastasis.
6. STC1 과발현 안정 세포의 조정 배지(CM)에서 확인된 분비성 STC1의 암 세포의 세포 성장, 침습, 및 이동을 유도 확인6. Confirmed secretory STC1 induced cell growth, invasion, and migration of cancer cells in conditioned medium (CM) of STC1-overexpressing stable cells
대조군 세포의 조정배지와 비교하여 STC1 과발현 안정 세포의 조정배지에서의 분비된 STC1 단백질의 양이 더 많은 것을 확인하였다(도 9a). 기능적 효과에 대해 세포 증식 및 콜로니 형성 검정을 실시하였으며, STC1 과발현 안정 세포의 조정배지를 방광암 세포에 처리했을 때 증식 및 콜로니 형성 능력이 상당히 가속화됨을 확인하였다(도 9a, b). 또한 세포 침입 및 이동 분석에서, STC1 과발현 안정 세포의 조정 배지에서 배양된 방광암 세포는 더 높은 세포 이동 및 침습 능력을 보였다(도 9c). 또한, 상처 회복 능력도 증가하였으며, STC1 과발현 안정 세포의 조정 배지를 농축하여 확인한 웨스턴 블롯팅 결과에서는 MMP1의 단백질 수준을 감소시켰으나 MMP2 및 9의 발현은 증가시켰으며, 이는 분비된 STC1 단백질이 방광암 세포에서 EMT 관련 유전자 발현을 촉진한다는 것 제시한다(도 9d). 또한, P15 세포의 조정 배지에서 분비된 STC1의 양은 P0 세포의 조정 배지에 보다 더 증가되었다(도 9e). 또한, 분비성 STC1 단백질의 양이 STC1 과발현 벡터를 형질주입한 세포와 STC1 과발현 안정 세포의 조정 배지에서 더 증가되었다(도 9e). 도 9f는, 도 8에서 진행한 실험의 마우스 혈액을 수집하여 혈청을 분리하고 혈청에 대해 인간 STC1 ELISA 검정을 실시한 결과를 나타낸 것으로, 대조군 세포를 주입한 마우스 그룹에 비해 STC1 과발현 안정 세포를 주입한 마우스 그룹의 혈청에서 더 많은 분비성 STC1 단백질이 발견되었다. STC1 분비 단백질이 방광암 세포에서 검출되는지를 확인하기 위해, P0 및 P15 세포의 조정 배지에 대해 인간 STC1 단백질의 ELISA 검정을 실시하였다. 즉, 분비된 STC1이 방광암 세포의 성장, 침습, 및 이동을 촉진한다.It was confirmed that the amount of secreted STC1 protein was greater in the conditioned medium of STC1 overexpressing stable cells compared to the conditioned medium of control cells (Figure 9a). Cell proliferation and colony formation assays were conducted for functional effects, and it was confirmed that when bladder cancer cells were treated with the conditioned medium from STC1-overexpressing stable cells, proliferation and colony formation abilities were significantly accelerated (Figures 9a, b). Additionally, in cell invasion and migration assays, bladder cancer cells cultured in conditioned medium from STC1-overexpressing stable cells showed higher cell migration and invasion abilities (Figure 9c). In addition, the wound recovery ability was also increased, and Western blotting results confirmed by concentrating the conditioned medium of STC1-overexpressing stable cells showed that the protein level of MMP1 was decreased, but the expression of MMP2 and 9 was increased, which means that the secreted STC1 protein is absorbed by bladder cancer cells. suggests that it promotes EMT-related gene expression (Figure 9d). Additionally, the amount of STC1 secreted in the conditioned medium of P15 cells was more increased than that in the conditioned medium of P0 cells (FIG. 9e). Additionally, the amount of secretory STC1 protein was further increased in the conditioned medium of cells transfected with the STC1 overexpression vector and STC1 overexpression stable cells (Figure 9e). Figure 9f shows the results of collecting mouse blood from the experiment conducted in Figure 8, separating serum, and performing a human STC1 ELISA assay on the serum. Compared to the mouse group injected with control cells, the group of mice injected with stable cells overexpressing STC1 was injected. More secretory STC1 protein was found in the serum of the mouse group. To determine whether STC1 secreted protein was detected in bladder cancer cells, an ELISA assay of human STC1 protein was performed on conditioned media from P0 and P15 cells. That is, secreted STC1 promotes the growth, invasion, and migration of bladder cancer cells.
7. 방광암 환자의 혈청 및 소변에서 확인된 분비성 STC1 단백질을 통해 방광암 환자의 진단 및 예후 예측 가능성 확인7. Confirmation of the possibility of diagnosing and predicting prognosis of bladder cancer patients through the secretory STC1 protein identified in the serum and urine of bladder cancer patients.
본 발명에서 분비된 STC1 단백질이 방광암의 전이와 관련됨을 확인하였다. 방광암 세포의 조정배지에서 분비된 STC1 단백질을 확인하고, 효과를 확인하기 위해, P0 및 P15 세포에 0, 100, 및 200 ng/mL의 재조합 인간 STC1 (rhSTC1) 단백질을 각각 0, 24, 48, 및 72시간 동안 처리하였다. rhSTC1 처리에 의한 세포 생존력은 72시간에 P0 세포에서 rhSTC1 처리에 의해 증가되었다. 그러나, P15 세포에는 효과가 없었다(도 10a). 방광암 세포에서 rhSTC1를 처리하여 확인한 세포 증식 능력도 100, 200 ng/mL rhSTC1가 처리된 P0 세포에서 콜로니 형성 능력 및 세포 이동 및 침습 능력 또한 증가하였다(도 10b). 이후, STC1 항체로 rhSTC1을 차단하여, rhSTC1의 전이 효과가 방광암 세포에서 없어지는 것을 확인하였다(도 10c). rhSTC1을 세포에 처리한 후에, 확인한 phosphorylated-FAK (p-FAK)의 발현이 증가하였다(도 10c). In the present invention, it was confirmed that the secreted STC1 protein is related to the metastasis of bladder cancer. To identify the STC1 protein secreted in the conditioned medium of bladder cancer cells and confirm the effect, 0, 100, and 200 ng/mL of recombinant human STC1 (rhSTC1) protein was added to P0 and P15 cells, respectively. and treated for 72 hours. Cell viability was increased by rhSTC1 treatment in P0 cells at 72 hours. However, there was no effect on P15 cells (Figure 10a). Cell proliferation ability, confirmed by treating bladder cancer cells with rhSTC1, also increased colony formation ability and cell migration and invasion ability in P0 cells treated with 100 and 200 ng/mL rhSTC1 (FIG. 10b). Afterwards, by blocking rhSTC1 with STC1 antibody, it was confirmed that the metastatic effect of rhSTC1 was lost in bladder cancer cells (FIG. 10c). After treating cells with rhSTC1, the expression of phosphorylated-FAK (p-FAK) increased (Figure 10c).
방광암 환자의 진단 및 예후를 예상하기 위한 STC1의 바이오마커로서의 유효성을 확인하기 위해, 건강한 대조 환자 및 방광암 환자의 혈청 및 소변에서 STC1의 발현을 확인하였다(도 11). 분비성 STC1의 증가된 수준이 건강한 대조 환자들에 비해 방광암 환자의 혈청에서 검출되었다(도 11a). 분비된 STC1 검출 또한 대조군 환자들에 비해 방광암 환자의 소변에서 확인되었으며, 환자의 등급 및 T 단계에 따라 달라지는 경향을 확인하였다. 재발이 확인된 샘플에서는 차이를 나타내지 않았다(도 11b). 이러한 결과들은 방광암 환자의 소변 및 혈청에서 분비된 STC1 단백질로 방광암을 확인할 수 있음을 제시한다. 이러한 결과들은 STC1가 바이오마커 후보로서 활용될 수 있음을 확인해 주었다.To confirm the effectiveness of STC1 as a biomarker for predicting the diagnosis and prognosis of bladder cancer patients, the expression of STC1 was confirmed in the serum and urine of healthy control patients and bladder cancer patients (FIG. 11). Increased levels of secretory STC1 were detected in the serum of bladder cancer patients compared to healthy control patients (Figure 11A). Detection of secreted STC1 was also confirmed in the urine of bladder cancer patients compared to control patients, and a tendency to vary depending on the patient's grade and T stage was confirmed. Samples with confirmed recurrence showed no differences (Figure 11B). These results suggest that bladder cancer can be identified using the STC1 protein secreted in the urine and serum of bladder cancer patients. These results confirmed that STC1 can be used as a biomarker candidate.
따라서, 본 발명의 방광암 바이오마커인 STC1은, 방광암 환자에서 고발현 되면, 환자의 불량한 예후와 관계된 것을 확인하였다. 또한, STC1의 발현이 증가할수록, 방광암 세포의 증식, 침윤 및 전이가 증가하는 것을 확인하였다. 또한, STC1이 방광암 환자의 혈청 또는 소변에서 검출되고, 환자의 임상 병기에 따른 발현 차이를 확인하여, 효과적으로 방광암의 진단 및 예후 예측에 이용될 수 있는 것을 확인하였다.Therefore, it was confirmed that STC1, the bladder cancer biomarker of the present invention, is associated with poor prognosis of patients when expressed at high levels in bladder cancer patients. In addition, it was confirmed that as the expression of STC1 increased, the proliferation, invasion, and metastasis of bladder cancer cells increased. In addition, it was confirmed that STC1 was detected in the serum or urine of bladder cancer patients, and that expression differences depending on the patient's clinical stage were confirmed, and that it can be effectively used to diagnose and predict the prognosis of bladder cancer.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.
<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> A novel biomarker that can predict the diagnosis and prognosis of bladder cancer in serum or urine <130> PN2206-329 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 12878 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens stanniocalcin 1 (STC1) gene sequence <400> 1 agtttgcaaa agccagaggt gcaagaagca gcgactgcag cagcagcagc agcagcggcg 60 gtggcagcag cagcagcagc ggcggcagca gcagcagcag cggaggcacc ggtggcagca 120 gcagcatcac cagcaacaac aacaaaaaaa aatcctcatc aaatcctcac ctaagctttc 180 agtgtatcca gatccacatc ttcactcaag ccaggagagg gaaagaggaa aggggggcag 240 gaaaaaaaaa aaacccaaca acttagcgga aacttctcag agaatgctcc aaaactcagc 300 agtgcttctg gtgctggtga tcagtgcttc tgcaacccat gaggcggagc agaatgactc 360 tgtgagcccc aggaaatccc gagtggcggc tcaaaactca ggtaagcagc aaacccaaga 420 gcggtttctc ccccaaagag ctgtcctcat ttgcctctcc cttttgcaac tgtgtctgtg 480 acggctgatc ttgataataa atgtgcttca tgcctgatgg caataaactg ccagtgtaat 540 ccaatagcct taggcagtgg agctcctttg tttaataaat tgcatgcaac taaacgaaga 600 agctggacgc tctgctgagg gtatttcatt gcataagcct agcctgattg cctgaaatct 660 ggcacgtacc ctcttggagg gggaggggtg agaggggagg aaaggcttga atgttggcat 720 gcttcaaagc gctctctata ctttccagaa gctgatctaa ggtaagacct ggcttgtttg 780 atgctgtccc tcttcctttc ctcttaagac tctacctctt cacttcttga cttctctacc 840 taagagaata cacagaaaag ctccctcttc agactgattt tcagaaccat agccctcaag 900 tctgacaaat ccagggcgga ttcagagaaa accctataca tcccaccccc aaccctgctg 960 cctgtctccc tcctggctct gtagggctcc attcagtcgt ccctgcagcc cgtagccaga 1020 gagcccctag aaaaaaaaaa ttgtgaaacg tagcagctgt ttctccaagg gtaaggtctc 1080 actttcgtct tgactcttgg agcagttaat tgtgcattat cttggcttct gaaggaaaag 1140 aatcattagg aagcctgcat tgacacctct gcctcttgac ttctctggct ccacttgttc 1200 cccttccaat actgccctct gtcctccaac tgtcagcaaa gaccggatca caggtgtcaa 1260 aatgctgcag atgatgatcc ccttggaaga gcacaggtcc ctgccgagag gagggctttt 1320 gcttcacttc cttttgtact tagttgctat agagatgcag tgattcacaa ttcgagcttg 1380 cctaaaacaa tagcagccct aatgcatatt aaaactgctt aaagcaaagt tataatttaa 1440 acccgtggaa atatataatt agggaaatga cttgtaacat cctagaggct gcgggtggtg 1500 ggagggggtg gggaagacta actgaaccat gcttttgatc tcagaggaga atcctatggc 1560 atgaaggctg cagatcattc cttgcctagc tgcctgccag aacacccccc ttctgtggga 1620 gaaaaattac ttttatggga gatcttatcc agaatgtaga aagcctcatg caagtttttt 1680 gcggttttct tccagtcttg catagggagg ccttggcctg gttattccac acctgtgttt 1740 cttctttctt tctttctttc tttctttctt ttttaaaagg cttttaatat gaaagagatt 1800 atgcatacct ctaagcactg tcctagtgga gggttggggt gaaacatgaa cggatcagat 1860 ttctttttgc caatatgacg gtaagttaaa agcctgaaat cagaagggtc tccaagtgga 1920 ctcctggcac agtcttctct gccatttaga ctaaaatccc atgcttttta ctcgggagtg 1980 catttttcca aagctagatg gattccctgg cccttttacc tcttaaacct gaccactagc 2040 cactgtctcc ttccttccca tgctttcaag tgctaagcaa tgacctttga ttctatggaa 2100 tggcttaaag gcactgtgca tttccagact tgagcctcac ttctccagac accaaaattg 2160 taggtaatgg tgaaacaggg agcgtgtgtc tgaaacagag aactccctcc ctgtccatca 2220 tgtactccag caatcataaa caaaaggagc ctgtctatcc tctctttctc aatcaaatgg 2280 gtctatcaat tgcccctcat gacagattcc taggataagt tcttctgacc tcatgtcctt 2340 gaaattcacc tctgcaccca cttgatccca cccagccatt cctcactgtt tgacccagaa 2400 tggatggatt tgtctctttc agctgaagtg gttcgttgcc tcaacagtgc tctacaggtc 2460 ggctgcgggg cttttgcatg cctggaaaac tccacctgtg acacagatgg gatgtatgac 2520 atctgtaaat ccttcttgta cagcgctgct aaatttgaca ctcaggtaat aaaaccttga 2580 cccctgctcc cactggctgc ttgtctttaa cagtgtgtta gaccatggtt cttaggaacc 2640 agtcttgtag ggaaccagcc ctgcttctca gcttccccac tctaaattcc catctcagca 2700 tgtgtatgca caagtgtgtg catgcacaca cacacacaca cacacacaca gaggcactca 2760 tctggccagg aaataggttc tgtataagac acgccccact cactctaatg catgattcta 2820 aggagctagt agaggctgtc ttataaggcc aggggtaata agccaaaaag attttcctct 2880 gtaagaggac ataaaaacgt gcatagagca aggaagttct ctctgcatga cctagcatac 2940 tagcttttga tctgaattct caaagtctca atctcaagaa tatgaagtca gattacgact 3000 atttttaagc tgcagaatgg ttgatcacgg ttcatctttt ttttctgtaa tttcatttcg 3060 gtagagctta attctcaagg gaatccaggg agatggggag agggcttttt tttttttcaa 3120 tccttagccg agacatgcaa tcttctcatt gcaaggatat gcagatggcc atgatacgtt 3180 gcccagatga gcccccttaa attcctccat gtatatgccc atcctctttg tcaggtcaag 3240 ctaaagacct ccctcttgtc cttcctccat ggcagggaaa agcattcgtc aaagagagct 3300 taaaatgcat cgccaacggg gtcacctcca aggtcttcct cgccattcgg aggtgctcca 3360 ctttccaaag gatgattgct gaggtgcagg aagagtgcta cagcaagctg aatgtgtgca 3420 gcatcgccaa gcggaaccct gaagccatca ctgaggtcgt ccagctgccc aatcacttct 3480 ccaacaggta caaactgagt ctactttttc acaatcaggg gactgggagc aggtggctgg 3540 ctggcagagc cagactggga gggagggtta aattgccaca gtcatgctct tattttagct 3600 tgcagtcttc ccagcttttg caggaaaaaa atatgtttgt gattggttgt gacagtttaa 3660 gcagcttggg cttttttttc cctactgtta taggaaaagt cttttcatgc agctcataga 3720 aagccagcct catctgcagc atccctgggt gctttcattg accatttttt gccataagcc 3780 tgaagggaat ggtgaagttt ctggaaacaa gcttttaaga catcagattt ggctgaaacc 3840 atgaccgtgc aacaacacaa ttggccatga ttaaaaaaaa aaaaaaaaaa aaaagccctc 3900 tggttaaatg tcataatgca gattttaagt ctccatctat ttcaaatcag tcccaaacac 3960 agtcagagat ttcactgggt agcccagagc aagccatctc acttccctgg ggctcagatt 4020 tcctttgtaa aaagcaaaat ggctggactg gaccattttc aggtttcctt ccaggcctta 4080 tgttatccca ttcctctagc ccaagatttc ctagaatgag tgtgttttat ggctgtccag 4140 atcccattct catcttagta ttcaaacacc acccaaggcc actgagctag tacagtaaca 4200 tctgggcatt gtacttttca gtgtttttac cagccaggag tgaattcctg ttacatgaga 4260 cagctggcat cctcagttca tgtgcgtggc ctggaaaggt tcccagcaag aactaagaga 4320 tgccaatggg gttggcgtgg aggtctggca ggggcagtgc agaccggtca agtttagccc 4380 cctgtgatat ccctcagcct aaaaacttga ctcccagcct tgaatttcta tgattctttg 4440 actctccact caaatctgca catatccaca atgatcagtg ctttgcatgt tactctgacc 4500 acctgagagt tatcacaggc ttatctgatt aatttcctag ccaggtgcca ccacccttct 4560 taagacagtg gcaacagcca agtccctctg gggcttacaa gtgatttagt catgtacata 4620 catgcaacaa aaaagtagtt cctccttagg gaaaggctaa ggtgcctaac acaaacgctg 4680 tgtaaaattt ctttttcacc cttaactaca ggtgagtgcc cttcagaacc acaaatcaat 4740 ttgtaaaaat cacttctcta gtccttggaa gtgactttat catggggttc taaatggttt 4800 cctcagaaca gaaaaatcca accattttct caagtgggct gtctctatat aactcttggt 4860 tttaaatttc ccctaggaag attcttattg cacagaggaa acaggtgcag agggtgggga 4920 ggggagatct gattaattat tggagacttc cttcaccatt aggaaaagaa atgaagaatt 4980 cttagccaga caggggctaa ggacaaagta tgtgtttgtg tggtagttag gggtgggggg 5040 agaggtacat tagcaaagcc actattacaa tttagatcct taaggattct gtgacaaatc 5100 atgatctata atacggcata tttccaattt tgaggcgata ctaatctccc tatttcatct 5160 gaagtagcat cttccattag aaaggcagag aggtctttag gcagaactat atagctactc 5220 gactagaggt cccaatttat ctaagaagag actagtaaat ttttaaccag gctattattt 5280 cttctaatca gtgccttatt ttcaacacct ttagaaaatg gtcacaaaga aagtcttctt 5340 atctccctct ttcatcttag ggactcatta ggaaaggtca attggagaaa aaaaaaatgt 5400 ggccagccct tgaaaaccgg aatcttccat gcaacctttc tccaaaaatt tcactttaac 5460 aggctatatt attcctgctc aggattgtag aaatgtccgg ttccatgctg ggcttgcttt 5520 gctccattga agctgctcac cttcctgaga catcctgctt tcccagcctc tatcccacca 5580 tagacacacg ctaagtcagt gccaattggc acagaattgc aagaggaagt agttacaggt 5640 tcattccacc ttcctgtgac ttatctagca tgagatttgg ggctgggaga atccaaaagc 5700 ctgtgactgg accaggagcc agaacaaggg gattttccat gacagaaatg cacctagagg 5760 gtaaagaggt agctggagtt tccacctgcc tgaatttgca gactatgctt tcaaccctgt 5820 gggaagcagg caaaattgat caggccagca gggcggtgcg aggcttggga accacttaga 5880 ccaacccaga tccagagtgg gaaaactgac aaagcttcca actctctagg gcatggatag 5940 ggaacaggaa acagaagaat aatattgatc aagggaaaca aaggactatt tataaaccct 6000 cacacagaac gttcctaggc agtatgtgtg tatttgggta agcatggctg aaacagtagc 6060 tatggagaat atctgatctt attctttatc aaaaaaaaaa aggtcaggga tggaattttc 6120 aaagtgagaa aaaagaaaca taattaaaaa ggtcctttcg ggctgggtga tcagaacact 6180 tagtccacca agtctttcca acacatctcc tccatcctcc ataaagataa agaaaggaat 6240 cttcttcttc tttttttttt tttttttttt tttttttgac agagtatcgc tctgttgccc 6300 aggctggagt acagtggtga gatctcagtt cactgcaacc tccatctcct gggttcaagc 6360 aattctcctg cctcagcctc cagagtagct gggattacaa gcactcacca ccacgcccag 6420 ctaatttttg tatttttagt agagacaggg tttcatcatg ttggccgggc tggtcttgaa 6480 ctcctgacct caggtgatcc acccatcttg gcctcccaaa gtgctgggac tgcaggcatg 6540 agccaccatg cccagccaag aaaggaatct tcttaagcag tcctaactac cactgcaaag 6600 gaggggatgt aagcagagct gctctatttt ttgtaaaatt gagaagaaaa atgtgtattg 6660 aatccctaga aacatagaat gtcatagttg gagatgactg ctgaggttaa ctagactaac 6720 tgcctcattt tactgattaa agaataaagg ctcagagagt ggagtgacct gctgaaggtc 6780 acacagcttg tgaataacta agttgggctt agaactcatg tggccttgag ccaagctatt 6840 tgatttctac ttcaaaaatg gcagccagca aaatctgcag gaaatagcca caacccaaaa 6900 atagaatacc aagcacaatt gaatatatgt gcaccgtggt agtccacaat taaatttgcg 6960 tcctttctct cccaaaagga tggacacatc ctaccaatag ctataggaac tataagacca 7020 tagcacaaga aggtgctcac taaataggtg acgattgcta agtcaactct tgctggagac 7080 acaacctctt aaataatcca tctcacttag tgacacagga aagggctatg tcattggcaa 7140 gcatgatccc agaggtaaat ggctattcca gcccttactg gacaggcact ggttaaccta 7200 ttgctcatac tgagaaactg gtgacatcaa ataatcctga caaattttcc agcctttgaa 7260 gaactactaa tggagacatg gactttgttt tatattctaa agagctttct cagcctgccc 7320 ttgctgagtt aacaaaagcc ccatgaggga aatagggtag attccagtgg gggtgggtgg 7380 ttctcactaa gagccctcag gctggcaagg gtcactgtca agaaacacca aggtcaggtg 7440 tcatatctga gccagcagac aaagcaacag taaaggtaga aaggtagaaa ccctccaaaa 7500 aagagcaggc actctgacaa agtggggcct gaaggaaatg tatggccctg aagctgcaaa 7560 gctggctgca gaagcttaat taccttaagg gtactagact tctttgtcca gacaagagct 7620 ttcaagtcct tctaaggaag aactagcatt tctttttatc cctttctagg gaaagattgt 7680 attttccagc ctatctgggg actcatctgg catgaacagc tctttctaga aatggaaggg 7740 ttaacttcta agttataaag tccttattgg agggatggga gatcagaaac cacccaccag 7800 gatctaatca ccctctgaaa tagacacagc catccaaaga cccaggtcct agcagagccc 7860 aagagccagg tgatgcctgg agatctatat gggtgtgtag atgtcagaag ctgctggtgc 7920 aaagttgttt attctatgtt tctccaaaac tggttttctc ctcatttccc catccaaagg 7980 agatctcctt tgactcatgt gctttgtaat tccaaagagt tggacaatgt tcctctgtgc 8040 cagagagtcc cagttttgac cacagctgga tataattctc cctggaggga agggtttcac 8100 caggcctgtg tcagcaacca agacactgca cttgcctctc agtccccagc ttccttctgc 8160 tgtgccacag aagggtgacc atatcacctc attacccacg ttccatgcta tttaccacac 8220 atctttgaaa taacctctct gggggattaa gttattctag aaagatgttt gatcctgtgc 8280 actgtttggt gagttaaaaa taaacaggtt tcttttcacg gaagacctgg tggccccaga 8340 gatccttggg ctcctctgag aaaaagttct ctccatgtaa atactgcagg tcactgtcac 8400 tgagtctctt attagcgaac acagaggtaa tgggttttga caactcaact gcgactacaa 8460 aatggaccaa atgcatccca gtctaaattg gataatacct gaactgtctt cccaaataac 8520 aggacaagtc attgatatgc cctaaatatt agacagttta atcctttctg agccacaaag 8580 cttagctaca ttgcttgggg gtatttaaag aattcgctgc aaagatgaag gtaactgata 8640 ccaaacacaa ggggttttgg taaaatttac taaaactaca gataagtttt cttctgggaa 8700 tcactactta ttggagagaa ttatattctt aaaactccat ttccttagtg gctgcaaaat 8760 aaatcaacaa atgtgcagtg atcaggcaat gtagcacgat ggcaccgggc cataaggaat 8820 acaaagaagg acacaaatcc tgccttcagt aagcttacag tctagtcagc gaaagaaagc 8880 catcacaaaa ccatcaaatg ctggcctatg tgatggcaaa caaagatcta taaatatcca 8940 aaaagggaag aggttagcaa actctggtat agtagaagaa ggattcatga attatagtag 9000 catgaaccct taaaaaccac caatatttcc atttaaatag acttcttgcc taggatttgg 9060 ccttctgact acaatatacc tttgaaggag ggctgaagat ggagtcagtc aatatctttt 9120 atcagggctg tccaagtatg tgataagggc attagatttt gtagaaaaat gcatggcaca 9180 cagaagaggg ggccttagac agaagtttca tcagaagatg cacagatctg catacacaca 9240 cacacacaca cacctgtgcc cacacaccac caaaaaaagg aaaattacaa aaaagcatca 9300 aaataattac acatgcatgt acaatggcta ctaatctcag tgatgagcat tggactcact 9360 tcattctaat ttgcttcacc tcatccacct tagtttcttc tctacaaaat gagagttgaa 9420 ctagtcacca aatggtctct gtggtcccag ctagctctgt aattctgttt ctaaaaatgt 9480 cagtcaccac agtcagcctg ctcttcaaag tgtgatctaa atctcctcaa ctctaaacat 9540 gaagatctgt cttgcattgt gtggtaaacc ctaggagcag atgcattgaa actagggtga 9600 catggcacaa gggttagaga gcaaaagcca ccttcagctg atgaactatt gatgtttgca 9660 tggtcaggcc attagggact cgtgttggct agagcccggg tgaaagcctg ggtctcgccg 9720 ggctctcact gggtgaccac catatgcact ctctttcttg atgcagatac tataacagac 9780 ttgtccgaag cctgctggaa tgtgatgaag acacagtcag cacaatcaga gacagcctga 9840 tggagaaaat tgggcctaac atggccagcc tcttccacat cctgcagaca gaccactgtg 9900 cccaaacaca cccacgagct gacttcaaca ggagacgcac caatgagccg cagaagctga 9960 aagtcctcct caggaacctc cgaggtgagg aggactctcc ctcccacatc aaacgcacat 10020 cccatgagag tgcataacca gggagaggtt attcacaacc tcaccaaact agtatcattt 10080 taggggtgtt gacacaccag ttttgagtgt actgtgcctg gtttgatttt tttaaagtag 10140 ttcctatttt ctatccccct taaagaaaat tgcatgaaac taggcttctg taatcaatat 10200 cccaacattc tgcaatggca gcattcccac caacaaaatc catgtgacca ttctgcctct 10260 cctcaggaga aagtaccctc ttttaccaac ttcctctgcc atgtttttcc cctgctcccc 10320 tgagaccacc cccaaacaca aaacattcat gtaactctcc agccattgta atttgaagat 10380 gtggatccct ttagaacggt tgccccagta gagttagctg ataaggaaac tttatttaaa 10440 tgcatgtctt aaatgctcat aaagatgtta aatggaattc gtgttatgaa tctgtgctgg 10500 ccatggacga atatgaatgt cacatttgaa ttcttgatct ctaatgagct agtgtcttat 10560 ggtcttgatc ctccaatgtc taattttctt tccgacacat ttaccaaatt gcttgagcct 10620 ggctgtccaa ccagactttg agcctgcatc ttcttgcatc taatgaaaaa caaaaagcta 10680 acatctttac gtactgtaac tgctcagagc tttaaaagta tctttaacaa ttgtcttaaa 10740 accagagaat cttaaggtct aactgtggaa tataaatagc tgaaaactaa tgtactgtac 10800 ataaattcca gaggactctg cttaaacaaa gcagtatata ataactttat tgcatataga 10860 tttagttttg taacttagct ttatttttct tttcctggga atggaataac tatctcactt 10920 ccagatatcc acataaatgc tccttgtggc cttttttata actaaggggg tagaagtagt 10980 tttaattcaa catcaaaact taagatgggc ctgtatgaga caggaaaaac caacaggttt 11040 atctgaagga ccccaggtaa gatgttaatc tcccagccca cctcaaccca gaggctactc 11100 ttgacttaga cctatactga aagatctctg tcacatccaa ctggaaattc caggaaccaa 11160 aaagagcatc cctatgggct tggaccactt acagtgtgat aaggcctact atacattagg 11220 aagtggcagt tctttactcg tcccctttca tcggtgcctg gtactctggc aaatgatgat 11280 ggggtgggag actttccatt aaatcaatca ggaatgagtc aatcagcctt taggtcttta 11340 gtccggggga cttggggctg agagagtata aataaccctg ggctgtccag ccttaataga 11400 cttctcttac attttcgtcc tgtagcacgc tgcctgccaa agtagtcctg gcagctggac 11460 catctctgta ggatcgtaaa aaaatagaaa aaaagaaaaa aaaaagaaag aaagagggaa 11520 aaagagctgg tggtttgatc atttctgcca tgatgtttac aagatggcga ccaccaaagt 11580 caaacgacta acctatctat gaacaacagt agtttctcag ggtcactgtc cttgaaccca 11640 acagtccctt atgagcgtca ctgcccacca aaggtcaatg tcaagagagg aagagaggga 11700 ggaggggtag gactgcaggg gccactccaa actcgcttag gtagaaacta ttggtgcttg 11760 actctcacta ggctaaactc aagatttgac caaatcgagt gatagggatc ctggtgggag 11820 gagagagggc acatctccag aaaaatgaaa agcaatacaa ctttaccata aagcctttaa 11880 aaccagtaac gtgctgctca aggaccaaga gcaattgcag cagacccagc agcagcagca 11940 gcagcacaaa cattgctgcc tttgtcccca cacagcctct aagcgtgctg acatcagatt 12000 gttaagggca tttttatact cagaactgtc ccatccccag gtccccaaac ttatggacac 12060 tgccttagcc tcttggaaat caggtagacc atattctaag ttagactctt cccctccctc 12120 ccacacttcc cacccccagg caaggctgac ttctctgaat cagaaaagct attaaagttt 12180 gtgtgttgtg tccattttgc aaacccaact aagccaggac cccaatgcga caagtagttc 12240 atgagtattc ctagcaaatt tctctctttc ttcagttcag tagatttcct tttttctttt 12300 cttttttttt tttttttttt ttggctgtga cctcttcaaa ccgtggtacc cccccttttc 12360 tccccacgat gatatctata tatgtatcta caatacatat atctacacat acagaaagaa 12420 gcagttctca caatgttgct agttttttgc ttctctttcc cccaccctac tccctccaat 12480 tcccccttaa acttccaaag cttcgtcttg tgtttgctgc agagtgattc gggggctgac 12540 ctagaccagt ttgcatgatt cttctcttgt gatttggttg cactttagac atttttgtgc 12600 cattatattt gcattatgta tttataattt aaatgatatt taggtttttg gctgagtact 12660 ggaataaaca gtgagcatat ctggtatatg tcattattta ttgttaaatt acatttttaa 12720 gctccatgtg catataaagg ttatgaaaca tatcatggta atgacagatg caagttattt 12780 tatttgctta tttttataat taaagatgcc atagcataat atgaagcctt tggtgaattc 12840 cttctaagat aaaaataata ataaagtgtt acgtttta 12878 <210> 2 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens stanniocalcin 1 (STC1) amino acid sequence <400> 2 Met Leu Gln Asn Ser Ala Val Leu Leu Val Leu Val Ile Ser Ala Ser 1 5 10 15 Ala Thr His Glu Ala Glu Gln Asn Asp Ser Val Ser Pro Arg Lys Ser 20 25 30 Arg Val Ala Ala Gln Asn Ser Ala Glu Val Val Arg Cys Leu Asn Ser 35 40 45 Ala Leu Gln Val Gly Cys Gly Ala Phe Ala Cys Leu Glu Asn Ser Thr 50 55 60 Cys Asp Thr Asp Gly Met Tyr Asp Ile Cys Lys Ser Phe Leu Tyr Ser 65 70 75 80 Ala Ala Lys Phe Asp Thr Gln Gly Lys Ala Phe Val Lys Glu Ser Leu 85 90 95 Lys Cys Ile Ala Asn Gly Val Thr Ser Lys Val Phe Leu Ala Ile Arg 100 105 110 Arg Cys Ser Thr Phe Gln Arg Met Ile Ala Glu Val Gln Glu Glu Cys 115 120 125 Tyr Ser Lys Leu Asn Val Cys Ser Ile Ala Lys Arg Asn Pro Glu Ala 130 135 140 Ile Thr Glu Val Val Gln Leu Pro Asn His Phe Ser Asn Arg Tyr Tyr 145 150 155 160 Asn Arg Leu Val Arg Ser Leu Leu Glu Cys Asp Glu Asp Thr Val Ser 165 170 175 Thr Ile Arg Asp Ser Leu Met Glu Lys Ile Gly Pro Asn Met Ala Ser 180 185 190 Leu Phe His Ile Leu Gln Thr Asp His Cys Ala Gln Thr His Pro Arg 195 200 205 Ala Asp Phe Asn Arg Arg Arg Thr Asn Glu Pro Gln Lys Leu Lys Val 210 215 220 Leu Leu Arg Asn Leu Arg Gly Glu Glu Asp Ser Pro Ser His Ile Lys 225 230 235 240 Arg Thr Ser His Glu Ser Ala 245 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> STC1_qRT PCR primer F <400> 3 agcgctgcta aatttgacac t 21 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> STC1_qRT PCR primer R <400> 4 ctttggaaag tggagcacct ccg 23 <110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> A novel biomarker that can predict the diagnosis and prognosis of bladder cancer in serum or urine <130> PN2206-329 <160> 4 <170> KoPatentIn 3.0 < 210> 1 <211> 12878 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens stanniocalcin 1 (STC1) gene sequence <400> 1 agtttgcaaa agccagaggt gcaagaagca gcgactgcag cagcagcagc agcagcggcg 60 gtggcagcag cag cagcagc ggcggcagca gcagcagcag cggaggcacc ggtggcagca 120 gcagcatcac cagcaacaac aacaaaaaaaa aatcctcatc aaatcctcac ctaagctttc 180 agtgtatcca gatccacatc ttcactcaag ccaggagagg gaaagaggaa aggggggcag 240 gaaaaaaaaa aaacccaaca acttagcgga aacttctcag agaatgctcc aaaactcagc 300 agtgcttctg gtgctggtga tcagtgcttc tgcaacccat gaggcggagc agaatgactc 360 tgtgagcccc aggaaatccc gagtggcggc tcaaaactca ggtaagcagc aaacccaaga 420 gcggtttctc ccccaaagag ctgtcctcat ttgcctctcc cttttgcaac tgtgtctgtg 48 0 acggctgatc ttgataataa atgtgcttca tgcctgatgg caataaactg ccagtgtaat 540 ccaatagcct taggcagtgg agctcctttg tttaataaat tgcatgcaac taaacgaaga 600 agctggacgc tctgctgagg gtatttcatt gcataagcct agcctgattg cctgaaatct 660 ggcacgtacc ctcttggagg gggaggggtg agaggggagg aaaggcttga atgttggcat 720 gcttcaaagc gctctctata ctttccagaa gctgatctaa ggtaagacct ggcttgtttg 780 atgctgtccc tcttcctttc ctcttaagac tctacctctt cacttcttga cttctctacc 840 taagagaata cacagaaaag ctccctcttc agactgattt tcagaaccat agccctcaag 900 tctgacaaat ccagggcgg a ttcagagaaa accctataca tcccaccccc aaccctgctg 960 cctgtctccc tcctggctct gtagggctcc attcagtcgt ccctgcagcc cgtagccaga 1020 gagcccctag aaaaaaaaaaa ttgtgaaacg tagcagctgt ttctccaagg gtaaggtctc 1080 actttcgtct tgactcttgg agcagttaat tgtgcattat cttggcttct gaaggaaaag 1140 aatcattagg aagcctgcat tga cacctct gcctcttgac ttctctggct ccacttgttc 1200 cccttccaat actgccctct gtcctccaac tgtcagcaaa gaccggatca caggtgtcaa 1260 aatgctgcag atgatgatcc ccttggaaga gcacaggtcc ctgccgagag gagggctttt 1320 gcttcacttc cttttgtact tagttgctat agagatgcag tgattcacaa ttcgagcttg 1380 cctaaaacaa tagcagccct aatgcatatt aaaactgctt aaagcaaagt tataatttaa 1440 acccgtggaa atatataatt agggaaatga cttgtaacat cctagaggct gcgggtggtg 1500 ggagggggtg gggaagacta actgaaccat gcttttgatc tcagaggaga atcctatggc 1560 atgaaggctg cagatcattc cttgcctagc tgcctg ccag aacaccccc ttctgtggga 1620 gaaaaaattac ttttatggga gatcttatcc agaatgtaga aagcctcatg caagtttttt 1680 gcggttttct tccagtcttg catagggagg ccttggcctg gttattccac acctgtgttt 1740 cttctttctt tctttcttt c tttctttctt ttttaaaagg cttttaatat gaaagagatt 1800 atgcatacct ctaagcactg tcctagtgga gggttggggt gaaacatgaa cggatcagat 1860 ttctttttgc caatatgacg gtaagttaaa agcctgaaat cagaagggtc tccaagtgga 1920 ctcctggcac agtcttctct gccatttaga ctaaaatccc atgcttttta ctcgggagtg 1980 catttttcca aagctagatg gattccctgg cccttttacc tcttaaacct gaccactagc 2040 cactgtctcc ttccttccca tgctttcaag tgctaagcaa tgacctttga ttctatggaa 2100 tggcttaaag gcactgtgca tttccagact tgagcctcac ttctccagac accaaaattg 2160 taggtaatgg tgaaacaggg agcgtgtgtc t gaaacagag aactccctcc ctgtccatca 2220 tgtactccag caatcataaa caaaaggagc ctgtctatcc tctctttctc aatcaaatgg 2280 gtctatcaat tgcccctcat gacagattcc taggataagt tcttctgacc tcatgtcctt 2340 gaaattcacc tctgcaccca cttgatccca cccagccatt cctcactgtt tgacccagaa 2400 tggatggatt tgtctctttc agctgaagtg gttcgttgcc tcaacagtgc t ctacaggtc 2460 ggctgcgggg cttttgcatg cctggaaaac tccacctgtg acacagatgg gatgtatgac 2520 atctgtaaat ccttcttgta cagcgctgct aaatttgaca ctcaggtaat aaaaccttga 2580 cccctgctcc cactggctgc ttgtctttaa cagtgtg tta gaccatggtt cttaggaacc 2640 agtcttgtag ggaaccagcc ctgcttctca gcttccccac tctaaattcc catctcagca 2700 tgtgtatgca caagtgtgtg catgcacaca cacacacaca cacacacaca gaggcactca 2760 tctggccagg aaataggttc tgtataagac acgccccact cactctaatg catgattcta 2820 aggagctagt agaggctgtc ttataaggcc aggggtaata agccaaaaag attttcctct 2880 gtaagaggac ataaaaacgt gcatagagca aggaagttct ctctgcatga cctagcatac 2940 tagcttttga tctgaattct caaagtctca atctcaagaa tatgaagtca gattacgact 3000 atttttaagc tgcagaatgg ttgatcacgg ttcatctttt ttttctgtaa tttcatttcg 3060 g tagagctta attctcaagg gaatccaggg agatggggag agggcttttt tttttttcaa 3120 tccttagccg agacatgcaa tcttctcatt gcaaggatat gcagatggcc atgatacgtt 3180 gcccagatga gcccccttaa attcctccat gtatatgccc atcctctttg tcaggtcaag 3240 ctaaagacct ccctcttgtc cttcctccat ggcagggaaa agcattcgtc aaagagagct 330 0 taaaatgcat cgccaacggg gtcacctcca aggtcttcct cgccattcgg aggtgctcca 3360 ctttccaaag gatgattgct gaggtgcagg aagagtgcta cagcaagctg aatgtgtgca 3420 gcatcgccaa gcggaaccct gaagccatca ctgaggtcgt ccagctgccc aat cacttct 3480 ccaacaggta caaactgagt ctactttttc acaatcaggg gactgggagc aggtggctgg 3540 ctggcagagc cagactggga gggagggtta aattgccaca gtcatgctct tattttagct 3600 tgcagtcttc ccagcttttg caggaaaaaa atatgtttgt gattggttgt gacagtttaa 3660 gcagcttggg cttttttttc cctactgtta taggaaaagt cttttcatgc agctcataga 3720 aagccagcc t catctgcagc atccctgggt gctttcattg accatttttt gccataagcc 3780 tgaagggaat ggtgaagttt ctggaaacaa gcttttaaga catcagattt ggctgaaacc 3840 atgaccgtgc aacaacacaa ttggccatga ttaaaaaaaaa aaaaaaaaaa aaaagccctc 3900 tggttaaat g tcataatgca gattttaagt ctccatctat ttcaaatcag tcccaaacac 3960 agtcagagat ttcactgggt agcccagagc aagccatctc acttccctgg ggctcagatt 4020 tcctttgtaa aaagcaaaat ggctggactg gaccattttc aggtttcctt ccaggcctta 4080 tgttatccca ttcctctagc ccaagatttc ctagaatgag tgtgttttat ggctgtccag 4140 atcccattct catcttagta ttca aacacc acccaaggcc actgagctag tacagtaaca 4200 tctgggcatt gtacttttca gtgtttttac cagccaggag tgaattcctg ttacatgaga 4260 cagctggcat cctcagttca tgtgcgtggc ctggaaaaggt tcccagcaag aactaagaga 4320 tgccaatggg gttggcg tgg aggtctggca ggggcagtgc agaccggtca agtttagccc 4380 cctgtgatat ccctcagcct aaaaacttga ctcccagcct tgaatttcta tgattctttg 4440 actctccact caaatctgca catatccaca atgatcagtg ctttgcatgt tactctgacc 4500 acctgagagt tatcacaggc ttatctgatt aatttcctag ccaggtgcca ccacccttct 4560 taagacagtg gcaacagcca agtccctctg gggcttaca a gtgatttagt catgtacata 4620 catgcaacaa aaaagtagtt cctccttagg gaaaggctaa ggtgcctaac acaaacgctg 4680 tgtaaaattt ctttttcacc cttaactaca ggtgagtgcc cttcagaacc acaaatcaat 4740 ttgtaaaaat cacttctcta gtccttggaa gtg actttat catggggttc taaatggttt 4800 cctcagaaca gaaaaaatcca accattttct caagtgggct gtctctatat aactcttggt 4860 tttaaatttc ccctaggaag attcttattg cacagaggaa acaggtgcag agggtgggga 4920 ggggagatct gattaattat tggagacttc cttcaccatt aggaaaagaa atgaagaatt 4980 cttagccaga caggggctaa ggacaaagta tgtgtttgtg tggtagttag gggtgg gggg 5040 agaggtacat tagcaaagcc actattacaa tttagatcct taaggattct gtgacaaatc 5100 atgatctata atacggcata tttccaattt tgaggcgata ctaatctccc tatttcatct 5160 gaagtagcat cttccattag aaaggcagag aggtctttag gcagaactat atagctactc 5220 gactagaggt cccaatttat ctaagaagag actagtaaat ttttaaccag gctattattt 5280 cttctaatca gtgccttatt ttcaacacct ttagaaaatg gtcacaaaga aagtcttctt 5340 atctccctct ttcatcttag ggactcatta ggaaaggtca attggagaaa aaaaaaatgt 5400 ggccagccct tgaaaaccgg aatcttccat gcaacctttc tccaaaaatt tcactttaac 5 460 aggctatatt attcctgctc aggattgtag aaatgtccgg ttccatgctg ggcttgcttt 5520 gctccattga agctgctcac cttcctgaga catcctgctt tcccagcctc tatcccacca 5580 tagacacacg ctaagtcagt gccaattggc acagaattgc aagaggaagt agttacaggt 5640 tcattccacc ttcctgtgac ttatctagca tgagatttgg ggctggggaga atccaaaagc 5700 ctgtgactgg accaggagcc agaacaaggg gattttccat gacagaaatg cacctagagg 5760 gtaaagaggt agctggagtt tccacctgcc tgaatttgca gactatgctt tcaaccctgt 5820 gggaagcagg caaaattgat caggccagca gggcggtgcg aggcttggga accacttaga 5880 c caacccaga tccagagtgg gaaaactgac aaagcttcca actctctagg gcatggatag 5940 ggaacaggaa acagaagaat aatattgatc aagggaaaca aaggactatt tataaaccct 6000 cacacagaac gttcctaggc agtatgtgtg tatttgggta agcatggctg aaacagtagc 6060 tatggagaat atctgatctt attctttatc aaaaaaaaaaa aggtcaggga tggaattttc 6120 aaagtgagaa aaaagaaaca taattaaaaa ggtcctttcg ggctgggtga tcagaacact 6180 tagtccacca agtctttcca acacatctcc tccatcctcc ataaagataa agaaaggaat 6240 cttcttcttc tttttttttt tttttttttt tttttttttttttttttgac agagtatcgc tctgtt gccc 6300 aggctggagt acagtggtga gatctcagtt cactgcaacc tccatctcct gggttcaagc 6360 aattctcctg cctcagcctc cagagtagct gggattacaa gcactcacca ccacgcccag 6420 ctaatttttg tatttttagt agagacaggg tttcatcatg ttggccgggc t ggtcttgaa 6480 ctcctgacct caggtgatcc acccatcttg gcctcccaaa gtgctgggac tgcaggcatg 6540 agccaccatg cccagccaag aaaggaatct tcttaagcag tcctaactac cactgcaaag 6600 gaggggatgt aagcagagct gctctatttt ttgtaaaatt gagaagaaaa atgtgtattg 6660 aatccctaga aacatagaat gtcatagttg gagatgactg ctgaggttaa ctagactaac 6720 tgcctcattt tactgattaa agaataaagg ctcagagagt ggagtgacct gctgaaggtc 6780 acacagcttg tgaataacta agttgggctt agaactcatg tggccttgag ccaagctatt 6840 tgatttctac ttcaaaaatg gcagccagca aaatctgcag gaaatagcca caacccaaaa 6900 atagaatacc aagcacaatt gaatatatg t gcaccgtggt agtccacaat taaatttgcg 6960 tcctttctct cccaaaagga tggacacatc ctaccaatag ctataggaac tataagacca 7020 tagcacaaga aggtgctcac taaataggtg acgattgcta agtcaactct tgctggagac 7080 acaacctctt aaataatcca tctcacttag tgacacagga aagggctatg tcattggcaa 7140 gcatgatccc agaggtaaat ggctattcca gcccttactg gacaggcact ggttaaccta 7200 ttgctcatac tgagaaactg gtgacatcaa ataatcctga caaattttcc agcctttgaa 7260 gaactactaa tggagacatg gactttgttt tatattctaa agagctttct cagcctgccc 7320 ttgctgagtt aacaaaagcc ccatgaggga aataggg tag attccagtgg gggtgggtgg 7380 ttctcactaa gagccctcag gctggcaagg gtcactgtca agaaacacca aggtcaggtg 7440 tcatatctga gccagcagac aaagcaacag taaaggtaga aaggtagaaa ccctccaaaa 7500 aagagcaggc actctgacaa agtggggcct gaaggaaatg tatggccctg aagctgcaaa 7560 gctggctgca gaagcttaat taccttaagg gtactagact tcttt gtcca gacaagagct 7620 ttcaagtcct tctaaggaag aactagcatt tctttttatc cctttctagg gaaagattgt 7680 attttccagc ctatctgggg actcatctgg catgaacagc tctttctaga aatggaaggg 7740 ttaacttcta agttataaag tccttattgg agggatggga gatca gaaac cacccaccag 7800 gatctaatca ccctctgaaa tagacacagc catccaaaga cccaggtcct agcagagccc 7860 aagagccagg tgatgcctgg agatctatat gggtgtgtag atgtcagaag ctgctggtgc 7920 aaagttgttt attctatgtt tctccaaaac tggttttctc ctcatttccc catccaaagg 7980 agatctcctt tgactcatgt gctttgtaat tccaaagagt tggacaatgt tcctctgtgc 8040 cagagagtcc cagttttgac cacagctgga tataattctc cctggaggga agggtttcac 8100 caggcctgtg tcagcaacca agacactgca cttgcctctc agtccccagc ttccttctgc 8160 tgtgccacag aag ggtgacc atatcacctc attacccacg ttccatgcta tttaccacac 8220 atctttgaaa taacctctct gggggattaa gttattctag aaagatgttt gatcctgtgc 8280 actgtttggt gagttaaaaa taaacaggtt tcttttcacg gaagacctgg tggccccaga 8340 gatccttggg ctcctctgag aaaaagttct ctccatgtaa atactgcagg tcactgtcac 8400 tgagtctctt attagcgaac acagaggtaa tgggttttga caactcaact gcg actacaa 8460 aatggaccaa atgcatccca gtctaaattg gataatacct gaactgtctt cccaaataac 8520 aggacaagtc attgatatgc cctaaatatt agacagttta atcctttctg agccacaaag 8580 cttagctaca ttgcttgggg gtatttaaag aattcgctgc aaagatgaag gta actgata 8640 ccaaacacaa ggggttttgg taaaatttac taaaactaca gataagtttt cttctgggaa 8700 tcactactta ttggagagaa ttatattctt aaaactccat ttccttagtg gctgcaaaat 8760 aaatcaacaa atgtgcagtg atcaggcaat gtagcacgat ggcaccgggc cataaggaat 8820 acaaagaagg acacaaatcc tgccttcagt aagcttacag tctagtcagc gaaagaaagc 8 880 catcacaaaa ccatcaaatg ctggcctatg tgatggcaaa caaagatcta taaatatcca 8940 aaaagggaag aggttagcaa actctggtat agtagaagaa ggattcatga attatagtag 9000 catgaaccct taaaaaccac caatatttcc atttaaatag acttcttgcc taggatttgg 9060 ccttctgact acaata tacc tttgaaggag ggctgaagat ggagtcagtc aatatctttt 9120 atcagggctg tccaagtatg tgataagggc attagatttt gtagaaaaat gcatggcaca 9180 cagaagaggg ggccttagac agaagtttca tcagaagatg cacagatctg catacacaca 9240 cacacacaca cacctgtgcc cacacaccac caaaaaaagg aaaattacaa aaaagcatca 9300 aaataattac acatgcatgt acaat ggcta ctaatctcag tgatgagcat tggactcact 9360 tcattctaat ttgcttcacc tcatccacct tagtttcttc tctacaaaat gagagttgaa 9420 ctagtcacca aatggtctct gtggtcccag ctagctctgt aattctgttt ctaaaaatgt 9480 cagtcaccac agtcagcctg ctcttcaaag tgtgatctaa atctcctcaa ctctaaacat 9540 gaagatctgt cttgcattgt gtggtaaacc ctaggagcag atgcattgaa actagggtga 9600 catggcacaa gggttagaga gcaaaagcca ccttcagctg atgaactatt gatgtttgca 9660 tggtcaggcc attagggact cgtgttggct agagcccggg tgaaagcctg ggtctcgccg 9720 ggctctcact gggtgacca c catatgcact ctctttcttg atgcagatac tataacagac 9780 ttgtccgaag cctgctggaa tgtgatgaag acacagtcag cacaatcaga gacagcctga 9840 tggagaaaat tgggcctaac atggccagcc tcttccacat cctgcagaca gaccactgtg 9900 cccaaacaca cccacgagct g acttcaaca ggagacgcac caatgagccg cagaagctga 9960 aagtcctcct caggaacctc cgaggtgagg aggactctcc ctcccacatc aaacgcacat 10020 cccatgagag tgcataacca gggagaggtt attcacaacc tcaccaaact agtatcattt 10080 taggggtgtt gacacaccag ttttgagtgt actgtgcctg gtttgatttt tttaaagtag 10140 ttcctatttt ctatccccct ta aagaaaat tgcatgaaac taggcttctg taatcaatat 10200 cccaacattc tgcaatggca gcattcccac caacaaaatc catgtgacca ttctgcctct 10260 cctcaggaga aagtaccctc ttttaccaac ttcctctgcc atgtttttcc cctgctcccc 10320 tgagaccacc cccaaacaca a aacattcat gtaactctcc agccattgta atttgaagat 10380 gtggatccct ttagaacggt tgccccagta gagttagctg ataaggaaac tttatttaaa 10440 tgcatgtctt aaatgctcat aaagatgtta aatggaattc gtgttatgaa tctgtgctgg 10500 ccatggacga atatgaatgt cacatttgaa ttcttgatct ctaatgagct agtgtcttat 10560 ggtcttgatc ctccaatgtc taattt tctt tccgacacat ttaccaaatt gcttgagcct 10620 ggctgtccaa ccagactttg agcctgcatc ttcttgcatc taatgaaaaa caaaaagcta 10680 acatctttac gtactgtaac tgctcagagc tttaaaagta tctttaacaa ttgtcttaaa 10740 accagagaat cttaaggtct aact gtggaa tataaatagc tgaaaactaa tgtactgtac 10800 ataaattcca gaggactctg cttaaacaaa gcagtatata ataactttat tgcatataga 10860 tttagttttg taacttagct ttatttttct tttcctggga atggaataac tatctcactt 10920 ccagatatcc acataaatgc tccttgtggc cttttttata actaaggggg tagaagtagt 10980 tttaattcaa catcaaaact taagatgggc ctgtatgaga caggaaaaac caacaggttt 11040 atctgaagga ccccaggtaa gatgttaatc tcccagccca cctcaaccca gaggctactc 11100 ttgacttaga cctatactga aagatctctg tcacatccaa ctggaaattc caggaaccaa 11160 aaagagcatc cctatgggct tggaccactt acagtgtgat aaggcctact atac attagg 11220 aagtggcagt tctttactcg tcccctttca tcggtgcctg gtactctggc aaatgatgat 11280 ggggtgggag actttccatt aaatcaatca ggaatgagtc aatcagcctt taggtcttta 11340 gtccggggga cttggggctg agagagtata aataaccctg ggctgtccag ccttaataga 11400 cttctcttac attttcgtcc tgtagcacgc tgcctgccaa agtag tcctg gcagctggac 11460 catctctgta ggatcgtaaa aaaatagaaa aaaagaaaaa aaaaagaaag aaagagggaa 11520 aaagagctgg tggtttgatc atttctgcca tgatgtttac aagatggcga ccaccaaagt 11580 caaacgacta acctatctat gaacaacagt agtt tctcag ggtcactgtc cttgaaccca 11640 acagtccctt atgagcgtca ctgcccacca aaggtcaatg tcaagagagg aagagaggga 11700 ggaggggtag gactgcaggg gccactccaa actcgcttag gtagaaacta ttggtgcttg 11760 actctcacta ggctaaactc aagatttgac caaatcgagt gatagggatc ctggtgggag 11820 gagagagggc acatctccag aaaaatgaaa agcaatacaa ctttaccata aagcctttaa 11880 aacca gtaac gtgctgctca aggaccaaga gcaattgcag cagacccagc agcagcagca 11940 gcagcacaaa cattgctgcc tttgtcccca cacagcctct aagcgtgctg acatcagatt 12000 gttaagggca tttttatact cagaactgtc ccatccccag gtccccaaac ttatggacac 12060 t gccttagcc tcttggaaat caggtagacc atattctaag ttagactctt cccctccctc 12120 ccacacttcc cacccccagg caaggctgac ttctctgaat cagaaaagct attaaagttt 12180 gtgtgttgtg tccattttgc aaacccaact aagccaggac cccaatgcga caagtagttc 12240 atgagtattc ctagcaaatt tctctctttc ttcagttcag tagatttcct tttttctttt 1 2300 ctttttttt tttttttttt ttggctgtga cctcttcaaa ccgtggtacc cccccttttc 12360 tccccacgat gatatctata tatgtatcta caatacatat atctacacat acagaaagaa 12420 gcagttctca caatgttgct agttttttgc ttctct ttcc cccaccctac tccctccaat 12480 tcccccttaa acttccaaag cttcgtcttg tgtttgctgc agagtgattc gggggctgac 12540 ctagaccagt ttgcatgatt cttctcttgt gatttggttg cactttagac atttttgtgc 12600 cattatattt gcattatgta tttataattt aaatgatatt taggtttttg gctgagtact 12660 ggaataaaca gtgagcatat ctggtatatg tcatttattta ttgttaaatt acatttttaa 12720 gctccatgt g catataaagg ttatgaaaca tatcatggta atgacagatg caagttattt 12780 tatttgctta tttttataat taaagatgcc atagcataat atgaagcctt tggtgaattc 12840 cttctaagat aaaaataata ataaagtgtt acgtttta 12878 <210> 2 <211> 247 <212 >PRT <213> Artificial Sequence <220> <223> Homo sapiens stanniocalcin 1 (STC1) amino acid sequence <400> 2 Met Leu Gln Asn Ser Ala Val Leu Leu Val Leu Val Ile Ser Ala Ser 1 5 10 15 Ala Thr His Glu Ala Glu Gln Asn Asp Ser Val Ser Pro Arg Lys Ser 20 25 30 Arg Val Ala Ala Gln Asn Ser Ala Glu Val Val Arg Cys Leu Asn Ser 35 40 45 Ala Leu Gln Val Gly Cys Gly Ala Phe Ala Cys Leu Glu Asn Ser Thr 50 55 60 Cys Asp Thr Asp Gly Met Tyr Asp Ile Cys Lys Ser Phe Leu Tyr Ser 65 70 75 80 Ala Ala Lys Phe Asp Thr Gln Gly Lys Ala Phe Val Lys Glu Ser Leu 85 90 95 Lys Cys Ile Ala Asn Gly Val Thr Ser Lys Val Phe Leu Ala Ile Arg 100 105 110 Arg Cys Ser Thr Phe Gln Arg Met Ile Ala Glu Val Gln Glu Glu Cys 115 120 125 Tyr Ser Lys Leu Asn Val Cys Ser Ile Ala Lys Arg Asn Pro Glu Ala 130 135 140 Ile Thr Glu Val Val Gln Leu Pro Asn His Phe Ser Asn Arg Tyr Tyr 145 150 155 160 Asn Arg Leu Val Arg Ser Leu Leu Glu Cys Asp Glu Asp Thr Val Ser 165 170 175 Thr Ile Arg Asp Ser Leu Met Glu Lys Ile Gly Pro Asn Met Ala Ser 180 185 190 Leu Phe His Ile Leu Gln Thr Asp His Cys Ala Gln Thr His Pro Arg 195 200 205 Ala Asp Phe Asn Arg Arg Arg Thr Asn Glu Pro Gln Lys Leu Lys Val 210 215 220 Leu Leu Arg Asn Leu Arg Gly Glu Glu Asp Ser Pro Ser His Ile Lys 225 230 235 240 Arg Thr Ser His Glu Ser Ala 245 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220 > <223> STC1_qRT PCR primer F <400> 3 agcgctgcta aatttgacac t 21 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> STC1_qRT PCR primer R<400> 4 ctttggaaag tggagcacct ccg 23
Claims (15)
상기 STC1은 서열번호 1의 염기서열을 포함하는 것인, 조성물.According to clause 2,
The composition wherein the STC1 includes the base sequence of SEQ ID NO: 1.
상기 STC1의 발현이 대조군의 기준치와 비교하여 증가되면, 암세포의 성장, 침습(invasion) 또는 이동(migration)이 증가되는 것인, 조성물.According to clause 2,
When the expression of STC1 is increased compared to the baseline value of the control group, the composition increases the growth, invasion, or migration of cancer cells.
상기 STC1의 발현이 대조군의 기준치와 비교하여 증가되면, 암의 임상 병기(clinical stage)가 증가되는 것인, 조성물.According to clause 2,
A composition wherein when the expression of STC1 is increased compared to the baseline value of the control group, the clinical stage of cancer is increased.
상기 STC1은 개체로부터 분리된 시료에서 측정되는 것인, 조성물.According to clause 2,
A composition wherein the STC1 is measured in a sample isolated from an individual.
상기 시료는 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 및 소변으로 이루어진 군에서 선택되는 것인, 조성물.According to clause 6,
The composition, wherein the sample is selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, and urine.
상기 분리된 생물학적 시료에서 STC1(Stanniocalcin-1)의 발현수준을 측정하는 단계; 및
상기 STC1의 발현 수준을 대조군의 기준치와 비교하는 단계;를 포함하는 방광암(bladder cancer)의 진단, 전이 또는 예후 예측을 위한 정보 제공 방법.isolating a biological sample from an individual;
Measuring the expression level of STC1 (Stanniocalcin-1) in the separated biological sample; and
A method of providing information for diagnosis, metastasis, or prognosis prediction of bladder cancer, comprising comparing the expression level of STC1 with a reference value of a control group.
상기 생물학적 시료는 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 및 소변으로 이루어진 군에서 선택되는 것인, 방법.According to clause 9,
The method wherein the biological sample is selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, and urine.
상기 STC1의 발현이 대조군의 기준치와 비교하여 증가되면, 방광암인 것으로 판단하는 것인, 방법.According to clause 9,
When the expression of STC1 increases compared to the baseline value of the control group, it is determined that it is bladder cancer.
상기 STC1의 발현이 대조군의 기준치와 비교하여 증가되면, 암세포의 성장, 침습(invasion) 또는 이동(migration)이 증가된 것으로 판단하는 것인, 방법.According to clause 9,
When the expression of STC1 is increased compared to the baseline value of the control group, it is determined that the growth, invasion, or migration of cancer cells is increased.
상기 분리된 생물학적 시료에 후보 물질을 처리하는 단계;
상기 후보물질이 처리된 생물학적 시료에서 STC1(Stanniocalcin-1)의 발현 수준을 측정하는 단계; 및
상기 STC1의 발현 수준을 대조군의 기준치와 비교하는 단계;를 포함하는 방광암(bladder cancer) 치료제의 스크리닝 방법.isolating a biological sample from an individual;
Processing the separated biological sample with a candidate substance;
Measuring the expression level of STC1 (Stanniocalcin-1) in the biological sample treated with the candidate material; and
A screening method for a treatment for bladder cancer comprising: comparing the expression level of STC1 with a reference value of a control group.
상기 STC1의 발현이 대조군의 기준치와 비교하여 저발현 되면, 항암효과가 있는 것으로 판단하는 것인, 방법.According to clause 14,
When the expression of STC1 is low compared to the reference value of the control group, it is determined that there is an anticancer effect.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/KR2022/008902 WO2022270926A1 (en) | 2021-06-22 | 2022-06-22 | Biomarker for diagnosing and predicting metastasis or prognosis of various cancers, and use thereof |
| KR1020220076394A KR20230175021A (en) | 2022-06-22 | 2022-06-22 | A novel biomarker that can predict the diagnosis and prognosis of bladder cancer in serum or urine |
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| Application Number | Priority Date | Filing Date | Title |
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| KR1020220076394A KR20230175021A (en) | 2022-06-22 | 2022-06-22 | A novel biomarker that can predict the diagnosis and prognosis of bladder cancer in serum or urine |
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| KR20230175021A true KR20230175021A (en) | 2023-12-29 |
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| KR1020220076394A KR20230175021A (en) | 2021-06-22 | 2022-06-22 | A novel biomarker that can predict the diagnosis and prognosis of bladder cancer in serum or urine |
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