KR20220163765A - Use of BUB1 as a biomarker for predicting the prognosis of Non-muscle invasive bladder cancer - Google Patents
Use of BUB1 as a biomarker for predicting the prognosis of Non-muscle invasive bladder cancer Download PDFInfo
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- KR20220163765A KR20220163765A KR1020210072335A KR20210072335A KR20220163765A KR 20220163765 A KR20220163765 A KR 20220163765A KR 1020210072335 A KR1020210072335 A KR 1020210072335A KR 20210072335 A KR20210072335 A KR 20210072335A KR 20220163765 A KR20220163765 A KR 20220163765A
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- bladder cancer
- invasive bladder
- bub1
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Abstract
Description
본 발명은 비근침윤성 방광암(Non-muscle Invasive Bladder Cancer; NMIBC)의 예후 예측을 위한 바이오마커 및 이의 용도에 관한 것으로, 자세하게는 비근침윤성 방광암의 예후 예측용 바이오마커; 상기 바이오마커를 이용한 비근침윤성 방광암의 예후 예측용 조성물; 상기 조성물을 포함하는 비근침윤성 방광암의 예후 예측용 키트 또는 마이크로어레이; 및 상기 바이오마커의 발현 수준을 측정하는 단계를 포함하는 비근침윤성 방광암의 예후 예측을 위한 정보를 제공하는 방법에 관한 것이다.The present invention relates to a biomarker for predicting the prognosis of non-muscle invasive bladder cancer (NMIBC) and its use, and specifically to a biomarker for predicting the prognosis of non-muscle invasive bladder cancer; A composition for predicting the prognosis of non-muscle invasive bladder cancer using the biomarker; A kit or microarray for predicting the prognosis of non-muscle invasive bladder cancer comprising the composition; And it relates to a method for providing information for predicting the prognosis of non-muscle invasive bladder cancer comprising measuring the expression level of the biomarker.
방광암(bladder cancer, BC)은 비뇨기계 영역에서 가장 빈번하게 발생하는 암으로서, 서양에서는 매년 인구 10만 명당 16.5명이 발병하는데 비하여 한국에서는 4.5명이 발생하는 것으로 보고되고 있다. 이처럼 서양에 비하여는 발생률이 낮으나, 해마다 발생률이 높아지고 있으며, 우리나라에서는 비뇨기계 암 중 가장 발생빈도가 높은 암으로 알려져 있다(Lee C, et al., 1992).Bladder cancer (BC) is the most frequently occurring cancer in the urinary system, and is reported to occur in Korea at 4.5 cases per 100,000 people per year in the West, compared to 16.5 cases per 100,000 people. As such, although the incidence rate is low compared to Western countries, the incidence rate is increasing every year, and in Korea, it is known as the most frequent cancer among urinary cancers (Lee C, et al., 1992).
방광암은 종양이 근육층을 침범했는지 여부에 따라 비근침윤성 방광암(Non-muscle Invasive Bladder Cancer; NMIBC)과 근침윤성 방광암(Muscle Invasive Bladder Cancer; MIBC)으로 분류된다. 비근침윤성 방광암은 암이 근육층의 침범 없이 점막에 국한된 병변으로써 경요도 방광절제술(transurethral resection of bladder tumor) 또는 방광내 항암제 또는 BCG를 주입함으로써 비교적 간단하게 치료가 가능하나, 암의 재발과 근침윤성 암으로의 진행이 문제가 된다. 한편, 근침윤성 방광암은 암이 근육층까지 침투한 상태를 말하는 것으로서, 이의 치료를 위하여는 근치적 방광적출술과 함께 복잡한 요로전환(urinary diversion)을 수행하여야 할 뿐 아니라, 환자에게 치명적인 결과를 초래할 수도 있다.Bladder cancer is classified into Non-muscle Invasive Bladder Cancer (NMIBC) and Muscle Invasive Bladder Cancer (MIBC) depending on whether the tumor has invaded the muscle layer. Non-muscle invasive bladder cancer is a lesion in which the cancer is confined to the mucous membrane without invasion of the muscle layer, and can be treated relatively simply by transurethral resection of bladder tumor or by injecting anticancer drugs or BCG into the bladder. However, cancer recurrence and muscle invasive cancer Progression is a problem. On the other hand, muscle-invasive bladder cancer refers to a state in which cancer has penetrated into the muscle layer, and for its treatment, not only must a radical cystectomy and complex urinary diversion be performed, but it can also cause fatal results to the patient. .
새로 진단된 방광암 환자의 약 75%가 비근침윤성 방광암이다(Burger et al., 2013). 비근침윤성 방광암 환자의 재발률은 60-70%, MIBC로의 진행률은 20-30%로, 이 암은 매우 이질적이기 때문에 환자들 사이에서 재발 및 진행 위험이 크다(Matulewicz and Steinberg, 2020). 근침윤성 방광암은 사망률이 높은 공격적인 암이다. 치료받지 않은 환자의 5년 생존율은 15% 미만이며, 표준 치료법인 근치 방광 절제술(radical cystectomy)을 시행한 후에도 환자의 약 50%가 사망한다. 새로운 치료법이 도입되었음에도 불구하고 MIBC 환자의 생존율은 지난 30년 동안 개선되지 않았다(Lobo et al., 2017). 따라서 비근침윤성 방광암 환자 중 근침윤성 방광암으로의 진행 가능성이 높은 환자를 미리 선별하여 보다 적극적인 치료를 시행하는 등 진행을 예방하는 것이 환자의 생존율 향상에 도움이 된다.About 75% of newly diagnosed bladder cancer patients have non-muscle invasive bladder cancer (Burger et al., 2013). Patients with non-muscular invasive bladder cancer have a recurrence rate of 60-70% and a progression rate of 20-30% to MIBC, and because this cancer is highly heterogeneous, there is a high risk of recurrence and progression among patients (Matulewicz and Steinberg, 2020). Muscular invasive bladder cancer is an aggressive cancer with a high mortality rate. The 5-year survival rate for untreated patients is less than 15%, and about 50% of patients die even after standard treatment, radical cystectomy. Despite the introduction of new treatments, the survival rate of patients with MIBC has not improved over the past 30 years (Lobo et al., 2017). Therefore, among patients with non-muscular invasive bladder cancer, it is helpful to improve the survival rate of patients to prevent progression, such as by pre-selecting patients with a high possibility of progression to muscle invasive bladder cancer and implementing more aggressive treatment.
이러한 배경 하에, 본 발명자들은 비근침윤성 방광암 환자 조직을 이용한 RNA 시퀀싱 분석 및 다양한 방광암 환자 코호트 분석을 통해 방광암 환자에서 차등 발현되는 유전자를 분석하고, 환자의 병리학적 특징 및 생존율과 BUB1 유전자의 발현량 사이의 연관성을 규명함으로써 본 발명을 완성하였다.Under this background, the present inventors analyzed genes differentially expressed in bladder cancer patients through RNA sequencing analysis using non-muscle invasive bladder cancer patient tissues and various bladder cancer patient cohort analyses, and analyzed the relationship between the patient's pathological characteristics and survival rate and the expression level of the BUB1 gene. The present invention was completed by identifying the correlation of.
따라서 본 발명의 목적은 비근침윤성 방광암의 예후를 효과적으로 예측할 수 있는 바이오마커를 제공하는 것이다.Accordingly, an object of the present invention is to provide a biomarker capable of effectively predicting the prognosis of non-muscle invasive bladder cancer.
본 발명의 다른 목적은 상기 바이오마커를 이용한 비근침윤성 방광암의 예후 예측용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for predicting the prognosis of non-muscle invasive bladder cancer using the biomarker.
본 발명의 다른 목적은, 상기 조성물을 포함하는 비근침윤성 방광암의 예후 예측용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for predicting the prognosis of non-muscle invasive bladder cancer comprising the composition.
본 발명의 또 다른 목적은, 상기 조성물을 포함하는 비근침윤성 방광암의 예후 예측용 마이크로어레이를 제공하는 것이다.Another object of the present invention is to provide a microarray for predicting the prognosis of non-muscle invasive bladder cancer comprising the composition.
본 발명의 또 다른 목적은, 상기 바이오마커를 이용하여 비근침윤성 방광암의 예후를 효과적으로 예측하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for effectively predicting the prognosis of non-muscle invasive bladder cancer using the biomarker.
상기와 같은 본 발명의 목적을 달성하기 위해서,In order to achieve the object of the present invention as described above,
본 발명은 BUB1 유전자를 유효성분으로 포함하는 비근침윤성 방광암의 예후 예측용 바이오마커 조성물을 제공한다.The present invention provides a biomarker composition for predicting the prognosis of non-muscle invasive bladder cancer comprising the BUB1 gene as an active ingredient.
본 발명의 일실시예에 있어서, 상기 바이오마커 조성물은 UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 및 SPAG5로 이루어진 군으로부터 선택되는 하나 이상의 유전자를 더 포함할 수 있다.In one embodiment of the present invention, the biomarker composition is UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 and SPAG5 It may further include one or more genes selected from the group consisting of
또한, 본 발명은 BUB1 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정하는 제제를 포함하는 비근침윤성 방광암의 예후 예측용 조성물을 제공한다.In addition, the present invention provides a composition for predicting the prognosis of non-muscle invasive bladder cancer comprising an agent for measuring the expression level of the mRNA of the BUB1 gene or the protein encoded by the gene.
본 발명의 일실시예에 있어서, 상기 조성물은 UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 및 SPAG5로 이루어진 군으로부터 선택되는 하나 이상의 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정하는 제제를 추가로 포함할 수 있다.In one embodiment of the present invention, the composition is a group consisting of UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 and SPAG5 It may further include an agent for measuring the expression level of the mRNA of one or more genes selected from or the protein encoded by the gene.
본 발명의 일실시예에 있어서, mRNA의 발현수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브이며; 단백질의 발현수준을 측정하는 제제는 선택된 유전자로부터 코딩되는 단백질에 특이적인 항체일 수 있다.In one embodiment of the present invention, the agent for measuring the expression level of mRNA is an antisense oligonucleotide, a primer pair or a probe that specifically binds to the gene; An agent for measuring the expression level of a protein may be an antibody specific for a protein encoded by a selected gene.
또한, 본 발명은 상기 조성물을 포함하는 비근침윤성 방광암의 예후 예측용 키트를 제공한다.In addition, the present invention provides a kit for predicting the prognosis of non-muscle invasive bladder cancer comprising the composition.
또한, 본 발명은 상기 조성물을 포함하는 비근침윤성 방광암의 예후 예측용 마이크로어레이를 제공한다.In addition, the present invention provides a microarray for predicting the prognosis of non-muscle invasive bladder cancer comprising the composition.
또한, 본 발명은 개체로부터 분리된 생물학적 시료에서 BUB1 유전자의 mRNA 발현수준 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정하는 단계를 포함하는, 비근침윤성 방광암의 예후 예측을 위한 정보를 제공하는 방법을 제공한다.In addition, the present invention provides information for predicting the prognosis of non-muscle invasive bladder cancer, comprising measuring the mRNA expression level of the BUB1 gene or the expression level of the protein encoded by the gene in a biological sample isolated from the subject. provides
본 발명의 일실시예에 있어서, 상기 단계는 UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 및 SPAG5로 이루어진 군으로부터 선택되는 하나 이상의 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 더 측정할 수 있다.of the present invention In one embodiment, the step is selected from the group consisting of UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 and SPAG5 The expression level of mRNA of one or more genes or proteins encoded by the genes can be further measured.
본 발명의 일실시예에 있어서, 상기 생물학적 시료는 환자의 조직, 세포, 혈액, 혈청, 혈장, 타액 및 소변으로 이루어진 군에서 선택될 수 있다.of the present invention In one embodiment, the biological sample may be selected from the group consisting of tissue, cell, blood, serum, plasma, saliva and urine of a patient.
본 발명은 RNA 시퀀싱 분석 및 다양한 방광암 환자 코호트(충북대학교 병원, GSE13507 데이터 세트 및 E-MTAT-4321 데이터 세트)에서 BUB1 유전자 발현 및 임상 결과와의 연관성 확인한 결과, 비근침윤성 방광암의 병기가 진행될수록 또는 조직학적 등급(세포분화도)이 나쁠수록 BUB1 유전자 발현이 높게 나타난바, 본 발명의 BUB1 유전자는 비근침윤성 방광암의 진행(progression) 및 재발(recurrence)을 포함하는 방광암의 예후 예측을 위한 마커로 유용하게 사용될 수 있다.The present invention confirmed the association between BUB1 gene expression and clinical results in RNA sequencing analysis and various bladder cancer patient cohorts (Chungbuk National University Hospital, GSE13507 data set and E-MTAT-4321 data set). As the histological grade (cell differentiation) was poor, the BUB1 gene expression was higher, so the BUB1 gene of the present invention is useful as a marker for predicting the prognosis of bladder cancer, including progression and recurrence of non-muscular invasive bladder cancer. can be used
도 1은 본 발명의 전체적인 연구 설계를 보여주는 흐름도이다.
도 2는 본 발명의 스크리닝 테스트에서 중복된 19개 유전자에 대한 유전자 온톨로지(GO) 분석 결과이다.
도 3은 RNA 시퀀싱 분석을 통해 얻은 비근침윤성 방광암(NMIBC) 조직과 정상 주변 조직(NAT)의 전사체 프로파일을 분석한 결과이다. 도 3a는 주성분 분석(Principal component analysis; PCA) 결과이며; 도 3b는 유전자 발현 히트맵(heatmap)이고; 도 3c는 비근침윤성 방광암(NMIBC)에서 크게 변경된 대표적인 유전자를 Volcano 플롯 그래프로 시각화한 것이며; 도 3d는 유전자 온톨로지(GO) 분석을 통해 방광암에서 상향 및 하향 조절된 유전자들이 관련된 pathway를 분석한 결과이며; 도 3e는 유전자세트증폭분석(GSEA) 결과이다(NES, normalized enrichment score).
도 4는 RNA-시퀀싱 분석에서 비근침윤성 방광암(NMIBC) 및 정상 주변 조직(NAT)에서 BUB1의 발현 정도를 확인한 결과이다.
도 5는 트레이닝 코호트(충북대학교 병원으로부터 제공받은 총 101개의 방광 조직 샘플, 표 2 참조)에서 BUB1 유전자의 발현을 RT-qPCR 분석을 통해 확인한 결과이다. 도 5a는 정상 방광 조직(Control)과 비근침윤성 방광암(NMIBC) 조직에서의 BUB1 유전자의 발현 수준을 비교한 결과이며; 도 5b는 비근침윤성 방광암 환자를 저등급(Low grade) 및 고등급(High grade) 군으로 나눈 후 BUB1 발현 수준을 비교한 결과이고; 도 5c는 비근침윤성 방광암(NMIBC) 환자가 나중에 근침윤성 방광암(MIBC)으로 진행된 경우와 진행되지 않은 경우로 나눈 후 BUB1 발현값을 비교한 결과이다.
도 6은 비근침윤성 방광암(NMIBC)의 무진행 생존(PFS)에 대한 BUB1의 효과를 보여주는 Kaplan-Meier 분석 결과를 나타낸 것이다. 도 6a는 비근침윤성 방광암(NMIBC) 환자를 BUB1의 중간 컷오프 값에 따라 두 그룹(BUB1 높음 및 BUB1 낮음)으로 분류한 후 무진행 생존율을 비교한 결과이고; 도 6b는 비근침윤성 방광암(NMIBC) 환자에서 종양 등급(저등급, 고등급)과 BUB1 발현 수준(BUB1 높음 및 BUB1 낮음)을 조합하여 4개의 그룹으로 분류한 후 무진행 생존율을 비교한 결과이다.
도 7은 GSE13507 코호트에서 BUB1 유전자 발현을 RT-qPCR 분석을 통해 확인한 결과이다. 도 7a는 정상 방광 조직(Control)과 비근침윤성 방광암(NMIBC) 조직에서의 BUB1 유전자의 발현 수준을 비교한 결과이며; 도 7b는 비근침윤성 방광암(NMIBC) 환자를 저등급(Low grade) 및 고등급(High grade) 군으로 나눈 후 BUB1 발현 수준을 비교한 결과이고; 도 7c는 비근침윤성 방광암(NMIBC) 환자를 Ta기 및 T1기 군으로 나눈 후 BUB1 발현 수준을 비교한 결과이며; 도 7d는 단변량 Cox 회귀 분석 결과 비근침윤성 방광암(NMIBC) 환자 중 BUB1 과발현 환자에서의 진행 위험비(Hazard ratio, HR)를 나타낸 것이며; 도 7e는 Kaplan Meier 생존 분석을 통해 비근침윤성 방광암(NMIBC) 환자의 BUB1 발현 수준에 따른 무진행 생존율을 비교한 결과이다.
도 8은 E-MTAB-4321 코호트에서 BUB1 유전자 발현을 RT-qPCR 분석을 통해 확인한 결과이다. 도 8a는 비근침윤성 방광암(NMIBC) 환자를 저등급(Low grade) 및 고등급(High grade) 군으로 나눈 후 BUB1 발현 수준을 비교한 결과이고; 도 8b는 비근침윤성 방광암(NMIBC) 환자를 Ta기 및 T1기 군으로 나눈 후 BUB1 발현 수준을 비교한 결과이며; 도 8c는 단변량 Cox 회귀 분석 결과 비근침윤성 방광암(NMIBC) 환자 중 BUB1 과발현 환자에서의 진행 위험비(Hazard ratio, HR)를 나타낸 것이며; 도 8d는 Kaplan Meier 생존 분석을 통해 비근침윤성 방광암(NMIBC) 환자를 BUB1 발현 수준에 따라 두 그룹(BUB1 높음 및 BUB1 낮음)으로 분류한 후 무진행 생존율을 비교한 결과이고; 도 8e는 Kaplan Meier 생존 분석을 통해 비근침윤성 방광암(NMIBC) 환자에서 병기(T1, Ta)와 BUB1 발현 수준(BUB1 높음 및 BUB1 낮음)을 조합하여 4개의 그룹으로 분류한 후 무진행 생존율을 비교한 결과이며; 도 8f는 Kaplan Meier 생존 분석을 통해 비근침윤성 방광암(NMIBC) 환자에서 종양 등급(저등급, 고등급)과 BUB1 발현 수준(BUB1 높음 및 BUB1 낮음)을 조합하여 4개의 그룹으로 분류한 후 무진행 생존율을 비교한 결과이다.
도 9는 두 가지 방광암 세포주(5637, T24)에서 siRNA 형질주입을 통해 BUB1 유전자를 넉다운(knockdown, KD)시킨 후, 방광암 세포의 변화를 관찰한 결과이다. 도 9a 및 9b는 두 세포주에서 BUB1 넉다운(KD)이 잘 되었는지 확인하기 위해 RT-qPCR을 통해 BUB1 발현 수준을 확인한 결과이며(9a: 5637, 9b: T24); 도 9c 및 9d는 유세포 분석을 이용한 세포 주기 분석을 통해 BUB1을 넉다운(KD)하지 않은 세포와 넉다운(KD)을 진행한 세포의 세포 주기 변화를 확인한 결과이다(9c: 5637, 9d: T24). * P <0.05, *** P <0.001, **** P <0.0001.1 is a flow chart showing the overall study design of the present invention.
2 is a Gene Ontology (GO) analysis result for 19 overlapping genes in the screening test of the present invention.
Figure 3 is a result of analyzing the transcriptome profile of non-muscle invasive bladder cancer (NMIBC) tissue and normal peripheral tissue (NAT) obtained through RNA sequencing analysis. 3A is a principal component analysis (PCA) result; 3B is a gene expression heatmap; Figure 3c is a Volcano plot graph visualization of representative genes significantly altered in non-muscle invasive bladder cancer (NMIBC); Figure 3d is a result of analyzing pathways related to up- and down-regulated genes in bladder cancer through gene ontology (GO) analysis; 3e is a gene set amplification analysis (GSEA) result (NES, normalized enrichment score).
Figure 4 is a result of confirming the expression level of BUB1 in non-muscle invasive bladder cancer (NMIBC) and normal peripheral tissue (NAT) in RNA-sequencing analysis.
Figure 5 shows the results of confirming the expression of the BUB1 gene in the training cohort (a total of 101 bladder tissue samples provided from Chungbuk National University Hospital, see Table 2) through RT-qPCR analysis. Figure 5a is a result of comparing the expression level of the BUB1 gene in normal bladder tissue (Control) and non-muscle invasive bladder cancer (NMIBC) tissue; Figure 5b is a result of comparing BUB1 expression levels after dividing patients with non-muscle invasive bladder cancer into low grade and high grade groups; Figure 5c is a result of comparison of BUB1 expression values after dividing non-muscular invasive bladder cancer (NMIBC) patients into those who later progressed to muscle invasive bladder cancer (MIBC) and those who did not.
Figure 6 shows the results of Kaplan-Meier analysis showing the effect of BUB1 on progression-free survival (PFS) of non-muscle invasive bladder cancer (NMIBC). Figure 6a shows the results of progression-free survival after classifying patients with non-muscle invasive bladder cancer (NMIBC) into two groups (BUB1 high and BUB1 low) according to the median cutoff value of BUB1; FIG. 6B is a result of comparing progression-free survival after classifying patients with non-muscle invasive bladder cancer (NMIBC) into 4 groups by combining tumor grade (low grade, high grade) and BUB1 expression level (BUB1 high and BUB1 low).
7 is a result of confirming BUB1 gene expression in the GSE13507 cohort through RT-qPCR analysis. Figure 7a is a result of comparing the expression level of the BUB1 gene in normal bladder tissue (Control) and non-muscle invasive bladder cancer (NMIBC) tissue; Figure 7b is a result of comparing BUB1 expression levels after dividing non-muscle invasive bladder cancer (NMIBC) patients into low grade and high grade groups; Figure 7c shows the results of comparison of BUB1 expression levels after dividing non-muscle invasive bladder cancer (NMIBC) patients into Stage Ta and Stage T1 groups; Figure 7d shows the Hazard ratio (HR) in non-muscle invasive bladder cancer (NMIBC) patients with BUB1 overexpression as a result of univariate Cox regression analysis; 7e is a result of comparing progression-free survival according to the BUB1 expression level of patients with non-muscle invasive bladder cancer (NMIBC) through Kaplan Meier survival analysis.
8 is a result of confirming BUB1 gene expression in the E-MTAB-4321 cohort through RT-qPCR analysis. Figure 8a is a result of comparing BUB1 expression levels after dividing non-muscle invasive bladder cancer (NMIBC) patients into low grade and high grade groups; Figure 8b is a result of comparing BUB1 expression levels after dividing non-muscle invasive bladder cancer (NMIBC) patients into Stage Ta and Stage T1 groups; Figure 8c shows the Hazard ratio (HR) in non-muscle invasive bladder cancer (NMIBC) patients with BUB1 overexpression as a result of univariate Cox regression analysis; Figure 8d is a result of comparing progression-free survival after classifying patients with non-muscle invasive bladder cancer (NMIBC) into two groups (BUB1 high and BUB1 low) according to the BUB1 expression level through Kaplan Meier survival analysis; Figure 8e compares progression-free survival rates after classifying into four groups by combining stages (T1, Ta) and BUB1 expression levels (BUB1 high and BUB1 low) in patients with non-muscle invasive bladder cancer (NMIBC) through Kaplan Meier survival analysis. result; 8f shows the progression-free survival rate after classifying into 4 groups by combining tumor grade (low grade, high grade) and BUB1 expression level (BUB1 high and BUB1 low) in patients with non-muscle invasive bladder cancer (NMIBC) through Kaplan Meier survival analysis. is the result of comparing
9 shows the results of observation of changes in bladder cancer cells after knockdown (KD) of the BUB1 gene in two bladder cancer cell lines (5637, T24) through siRNA transfection. Figures 9a and 9b show the results of confirming the BUB1 expression level through RT-qPCR to confirm that BUB1 knockdown (KD) was successful in the two cell lines (9a: 5637, 9b: T24); 9c and 9d show the results of confirming cell cycle changes in cells without BUB1 knockdown (KD) and cells after knockdown (KD) through cell cycle analysis using flow cytometry (9c: 5637, 9d: T24). *P < 0.05, ***P < 0.001, ****P < 0.0001.
하나의 양태로서, 본 발명은 BUB1 유전자의 비근침윤성 방광암의 예후 예측을 위한 바이오마커로서의 용도에 관한 것이다.In one aspect, the present invention relates to the use of the BUB1 gene as a biomarker for predicting the prognosis of non-muscle invasive bladder cancer.
본 발명의 일구체예에서, 비근침윤성 방광암의 예후 예측을 위한 바이오마커로 BUB1 유전자 이외에 UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 및 SPAG5로 이루어진 군으로부터 선택되는 하나 이상의 유전자를 더 포함할 수 있다.In one embodiment of the present invention, as a biomarker for predicting the prognosis of non-muscle invasive bladder cancer, in addition to the BUB1 gene, UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, It may further include one or more genes selected from the group consisting of TACC3, MCM2, RECQL4 and SPAG5.
본 발명의 상기 BUB1 유전자는 서열번호 1의 폴리뉴클레오티드 서열로 이루어질 수 있으며; 상기 UHRF1 유전자는 서열번호 2의 폴리뉴클레오티드 서열로 이루어질 수 있고; 상기 AURKB 유전자는 서열번호 3의 폴리뉴클레오티드 서열로 이루어질 수 있으며; 상기 TPX2 유전자는 서열번호 4의 폴리뉴클레오티드 서열로 이루어질 수 있고; 상기 BUB1B 유전자는 서열번호 5의 폴리뉴클레오티드 서열로 이루어질 수 있으며; 상기 KIF20A 유전자는 서열번호 6의 폴리뉴클레오티드 서열로 이루어질 수 있고; 상기 CDC20 유전자는 서열번호 7의 폴리뉴클레오티드 서열로 이루어질 수 있으며; 상기 TK1 유전자는 서열번호 8의 폴리뉴클레오티드 서열로 이루어질 수 있고; 상기 CEP55 유전자는 서열번호 9의 폴리뉴클레오티드 서열로 이루어질 수 있으며; 상기 TTK 유전자는 서열번호 10의 폴리뉴클레오티드 서열로 이루어질 수 있고; 상기 CCNB2 유전자는 서열번호 11의 폴리뉴클레오티드 서열로 이루어질 수 있으며; 상기 HJURP 유전자는 서열번호 12의 폴리뉴클레오티드 서열로 이루어질 수 있고; 상기 NUSAP1 유전자는 서열번호 13의 폴리뉴클레오티드 서열로 이루어질 수 있으며; 상기 TOP2A 유전자는 서열번호 14의 폴리뉴클레오티드 서열로 이루어질 수 있고; 상기 PRC1 유전자는 서열번호 15의 폴리뉴클레오티드 서열로 이루어질 수 있으며; 상기 TACC3 유전자는 서열번호 16의 폴리뉴클레오티드 서열로 이루어질 수 있고; 상기 MCM2 유전자는 서열번호 17의 폴리뉴클레오티드 서열로 이루어질 수 있으며; 상기 RECQL4 유전자는 서열번호 18의 폴리뉴클레오티드 서열로 이루어질 수 있고; 상기 SPAG5 유전자는 서열번호 19의 폴리뉴클레오티드 서열로 이루어질 수 있다.The BUB1 gene of the present invention may consist of the polynucleotide sequence of SEQ ID NO: 1; The UHRF1 gene may consist of the polynucleotide sequence of SEQ ID NO: 2; The AURKB gene may consist of the polynucleotide sequence of SEQ ID NO: 3; The TPX2 gene may consist of the polynucleotide sequence of SEQ ID NO: 4; The BUB1B gene may consist of the polynucleotide sequence of SEQ ID NO: 5; The KIF20A gene may consist of the polynucleotide sequence of SEQ ID NO: 6; The CDC20 gene may consist of the polynucleotide sequence of SEQ ID NO: 7; The TK1 gene may consist of the polynucleotide sequence of SEQ ID NO: 8; The CEP55 gene may consist of the polynucleotide sequence of SEQ ID NO: 9; The TTK gene may consist of the polynucleotide sequence of SEQ ID NO: 10; The CCNB2 gene may consist of the polynucleotide sequence of SEQ ID NO: 11; The HJURP gene may consist of the polynucleotide sequence of SEQ ID NO: 12; The NUSAP1 gene may consist of the polynucleotide sequence of SEQ ID NO: 13; The TOP2A gene may consist of the polynucleotide sequence of SEQ ID NO: 14; The PRC1 gene may consist of the polynucleotide sequence of SEQ ID NO: 15; The TACC3 gene may consist of the polynucleotide sequence of SEQ ID NO: 16; The MCM2 gene may consist of the polynucleotide sequence of SEQ ID NO: 17; The RECQL4 gene may consist of the polynucleotide sequence of SEQ ID NO: 18; The SPAG5 gene may consist of the polynucleotide sequence of SEQ ID NO: 19.
본 발명에 따른 비근침윤성 방광암의 예후 예측을 위한 마커 유전자로서 상기 선택된 유전자는 ⅰ) 충북대학교병원 RNA 시퀀싱에서 비근침윤성 방광암 환자 조직과 방광암이 포함되지 않은 정상 주변 조직 사이의 차등적으로 증가된 발현 수준을 나타내었으며; ⅱ) GSE13507 코호트에서 정상 대조군 대비 비근침윤성 방광암(NMIBC) 환자에서 차등적으로 증가된 발현 수준을 나타내었고; ⅲ) GSE13507 코호트에서 비근침윤성 방광암(NMIBC) 대비 근침윤성 방광암(MIBC) 환자에서 차등적으로 증가된 발현 수준을 나타내었다(도 1 다이아그램 참조).The gene selected as a marker gene for predicting the prognosis of non-muscle invasive bladder cancer according to the present invention is i) differentially increased expression level between non-muscle invasive bladder cancer patient tissue and normal surrounding tissue that does not contain bladder cancer in RNA sequencing at Chungbuk National University Hospital represents; ii) showed differentially increased expression levels in non-muscle invasive bladder cancer (NMIBC) patients compared to normal controls in the GSE13507 cohort; iii) In the GSE13507 cohort, expression levels were differentially increased in patients with muscle invasive bladder cancer (MIBC) compared to non-muscle invasive bladder cancer (NMIBC) (see diagram in FIG. 1).
또한, 본 발명의 비근침윤성 방광암의 예후 예측을 위한 마커 유전자로서 상기 선발된 19개의 유전자의 생물학적 특징을 유전자 온톨로지(GO) 분석을 통해 상기 유전자가 핵 분열, 방추 조직 및 세포질 분열과 같은 세포주기의 중요한 단계에 관련되어 있음을 확인하였다(도 2 참조). In addition, through gene ontology (GO) analysis of the biological characteristics of the 19 genes selected as marker genes for prognosis of non-muscle-invasive bladder cancer of the present invention, the genes are involved in cell cycle functions such as nuclear division, spindle organization, and cytokinesis. It was confirmed that it is involved in an important step (see Fig. 2).
또한, 상기 19개의 유전자 중 스핀들 체크 포인트 조절자(spindle checkpoint regulator)인 BUB1의 NMIBC 병인 및 예후에 미치는 역할을 추가적으로 평가하기 위하여, 비근침윤성 방광암(NMIBC) 조직에서 BUB1의 mRNA 발현을 확인한 결과 대조군(정상 방광 점막)보다 비근침윤성 방광암(NMIBC) 조직에서 상당히 과발현되었음을 확인하였으며(도 5a 참조), 비근침윤성 방광암(NMIBC) 환자에서 병기가 진행될수록 또는 조직학적 등급(세포분화도)이 나쁠수록 BUB1의 mRNA 발현이 증대되는 것을 확인하였다(도 5b 내지 5c 참조). In addition, in order to further evaluate the role of BUB1, a spindle checkpoint regulator among the 19 genes, on the pathogenesis and prognosis of NMIBC, the mRNA expression of BUB1 was confirmed in non-muscle invasive bladder cancer (NMIBC) tissue, and the control group ( It was confirmed that it was significantly overexpressed in non-muscle-invasive bladder cancer (NMIBC) tissues than normal bladder mucosa) (see FIG. 5a), and as the stage progressed or the histological grade (cell differentiation) worsened in non-muscle-invasive bladder cancer (NMIBC) patients, the mRNA of BUB1 increased. It was confirmed that expression was increased (see Figs. 5b to 5c).
또한, 비근침윤성 방광암(NMIBC)에서 BUB1의 예후력을 평가하기 위해 생존 분석을 수행한 결과, 상대적으로 BUB1 발현 수준이 낮은 환자 대비 BUB1 발현 수준이 높은 환자에서 무진행 생존율이 낮아지는 것으로 나타났으며(도 6a 참조), BUB1 발현 수준이 높으면서 조직학적 등급(세포분화도)이 나쁜 경우 무진행 생존율이 더욱 낮아지는 것을 확인하였다(도 6b 참조). In addition, as a result of survival analysis to evaluate the prognostic power of BUB1 in non-muscle invasive bladder cancer (NMIBC), it was found that the progression-free survival rate was lower in patients with high BUB1 expression levels compared to patients with relatively low BUB1 expression levels. (See Fig. 6a), it was confirmed that the progression-free survival rate was further lowered when the histological grade (cell differentiation degree) was poor while the BUB1 expression level was high (see Fig. 6b).
뿐만 아니라, 방광암 환자 코호트 데이터(GSE13507 및 E-MTAT-4321)를 사용하여 비근침윤성 방광암(NMIBC)에서 BUB1의 발현 수준을 검증한 결과, BUB1 mRNA 발현은 정상 대조군과 비교하여 비근침윤성 방광암(NMIBC)에서 유의하게 향상되고(도 7a 참조), 비근침윤성 방광암(NMIBC) 환자에서 병기가 진행될수록 또는 조직학적 등급(세포분화도)이 나쁠수록 BUB1의 발현량이 더 높아지는 것을 확인하였으며(도 7b, 7c, 8a 및 8b 참조), 이와 더불어 BUB1 발현 수준이 높은 경우 무진행 생존율이 낮아지는 것을 확인하였다(도 7e 및 8d 참조). In addition, as a result of verifying the expression level of BUB1 in non-muscle-invasive bladder cancer (NMIBC) using bladder cancer patient cohort data (GSE13507 and E-MTAT-4321), BUB1 mRNA expression was higher in non-muscle-invasive bladder cancer (NMIBC) compared to normal controls. was significantly improved (see FIG. 7a), and it was confirmed that the expression level of BUB1 increased as the stage progressed or the histological grade (cell differentiation degree) worsened in non-muscle invasive bladder cancer (NMIBC) patients (FIG. 7b, 7c, 8a and 8b), and in addition, it was confirmed that the progression-free survival rate was lowered when the BUB1 expression level was high (see FIGS. 7e and 8d).
특히, E-MTAT-4321 코호트에서 종양 단계 및 등급을 BUB1 발현과 통합할 때, BUB1의 발현이 높은 T1 단계 그룹의 비근침윤성 방광암(NMIBC) 환자는 최악의 무진행 생존을 보였고, BUB1의 발현이 낮은 Ta 단계 그룹의 비근침윤성 방광암(NMIBC) 환자에서 가장 만족스러운 무진행 생존(PFS)을 보여주었다(도 8e 참조). 그리고 BUB1 발현이 높은 고등급(High grade) 그룹의 비근침윤성 방광암(NMIBC) 환자는 가장 힘든 무진행 생존을 겪는 반면, BUB1 발현이 낮은 저등급(low grade) 그룹의 비근침윤성 방광암(NMIBC) 환자는 가장 낙관적인 무진행 생존을 보여주었다(도 8f 참조).In particular, when integrating tumor stage and grade with BUB1 expression in the E-MTAT-4321 cohort, patients with non-muscle-invasive bladder cancer (NMIBC) in the T1 stage group with high BUB1 expression showed the worst progression-free survival, Patients with non-muscle invasive bladder cancer (NMIBC) in the low Ta stage group showed the most satisfactory progression-free survival (PFS) (see Fig. 8e). In addition, non-muscle invasive bladder cancer (NMIBC) patients in the high grade group with high BUB1 expression experience the most difficult progression-free survival, whereas non-muscle invasive bladder cancer (NMIBC) patients in the low grade group with low BUB1 expression showed the most optimistic progression-free survival (see FIG. 8F ).
이에, 본 발명에 따른 비근침윤성 방광암의 예후 예측을 위한 마커로서 상기 선발된 유전자들은 비근침윤성 방광암의 재발 및 진행에 대한 중요한 지표가 되는 종양 단계(stage) 및 등급(grade) 사이에 발현 수준의 차이를 보이는 차등적으로 발현된 유전자들이다.Therefore, the genes selected as markers for predicting the prognosis of non-muscle-invasive bladder cancer according to the present invention show differences in expression levels between tumor stages and grades, which are important indicators for the recurrence and progression of non-muscle-invasive bladder cancer. are differentially expressed genes that show
그러므로 본 발명에 따른 상기 유전자의 발현수준을 측정함으로써 보다 정확하게 비근침윤성 방광암의 예후를 예측할 수 있다.Therefore, the prognosis of non-muscle invasive bladder cancer can be more accurately predicted by measuring the expression level of the gene according to the present invention.
본 발명에서 용어 "비근침윤성 방광암(Non-muscle Invasive Bladder Cancer; NMIBC)"이란, 암이 근육층의 침범없이 점막에 국한된 병변을 가지는 방광암으로서 '비근침습성 방광암' 또는 '표재성 방광암'이라고도 불린다.In the present invention, the term "Non-muscle Invasive Bladder Cancer (NMIBC)" is a bladder cancer in which the cancer has lesions confined to the mucous membrane without invasion of the muscle layer, and is also called 'non-muscle invasive bladder cancer' or 'superficial bladder cancer'.
본 발명에서 용어, "차등적으로 발현된 유전자(differentially expressed genes, DEGs)"는 정상 또는 대조군 시료에서의 발현에 비하여, 질병, 구체적으로 암, 예를 들어 방광암 환자에서 그의 발현이 보다 높거나 보다 낮은 수준으로 활성화되는 유전자를 말한다. 이 용어는 또한 그의 발현이 동일한 질병의 상이한 병기에서 보다 높거나 보다 낮은 수준으로 활성화되는 유전자를 포함한다. 차등적으로 발현되는 유전자는 핵산 수준 또는 단백질 수준에서 활성화되거나 또는 억제될 수 있거나, 교대 스플라이싱을 통해 상이한 폴리펩티드 산물을 생성시킬 수 있음을 또한 알 수 있다. 이러한 차이는, 예를 들면 폴리펩티드의 mRNA 수준, 표면 발현, 분비 또는 다른 분배에 있어서의 변화에 의해 입증될 수 있다. 차등적인 유전자 발현은 2개 이상의 유전자 또는 상기 유전자 산물간의 발현 비교, 또는 2개 이상의 유전자 또는 상기 유전자 산물간의 발현 비율의 비교, 또는 동일한 유전자의 2개의 상이하게 처리된 산물의 비교(이들은 정상 대상과 질병, 구체적으로 비근침윤성 방광암을 앓는 대상 사이에서 또는 동일한 질병의 다양한 병기 사이에서 다름)를 포함할 수 있다. 차등적인 발현은, 예를 들면 정상 세포와 질병에 걸린 세포들 사이에서, 또는 상이한 질병 사건 또는 질병 병기를 거치는 세포들 사이에서 유전자 또는 그의 발현 산물에서 일시적인 또는 세포 발현 양상의 정량적 및 정성적 차이를 모두 포함한다. 본 발명의 목적상, "차등적인 유전자 발현"은 질병에 걸린 대상의 질병 발생의 다양한 병기 및 등급에서 주어진 유전자의 발현 사이에 적어도 약 1.5배, 바람직하게는 적어도 약 2배, 보다 바람직하게는 적어도 약 4배의 차이가 있을 때 존재하는 것으로 간주된다.As used herein, the term "differentially expressed genes (DEGs)" refers to a higher or higher expression in patients with a disease, specifically cancer, for example, bladder cancer, compared to expression in a normal or control sample. A gene that is activated at a low level. The term also includes genes whose expression is activated at higher or lower levels in different stages of the same disease. It can also be seen that genes that are differentially expressed can be activated or inhibited at the nucleic acid level or protein level, or can result in different polypeptide products through alternating splicing. Such differences may be evidenced, for example, by changes in mRNA levels, surface expression, secretion or other distribution of the polypeptide. Differential gene expression is a comparison of expression between two or more genes or the product of said genes, or a comparison of the expression ratio between two or more genes or said gene products, or a comparison of two differently processed products of the same gene (which is different from a normal subject). different between subjects with a disease, specifically non-muscle invasive bladder cancer, or between various stages of the same disease). Differential expression is the quantitative and qualitative difference in temporal or cellular expression patterns in a gene or its expression product, for example, between normal and diseased cells, or between cells undergoing different disease events or stages of a disease. All inclusive. For purposes of the present invention, "differential gene expression" means at least about 1.5-fold, preferably at least about 2-fold, more preferably at least about 2-fold, more preferably at least about 1.5-fold, preferably at least about 2-fold, more preferably at least It is considered to be present when there is a difference of about 4 fold.
본 발명에서 용어, "예후"는 방광암과 같은 질환의, 예를 들어 재발(동일한 병리학적 단계의 원발성 NMIBC의 재발로 정의), 전이성 확산, 및 약물 내성을 비롯한 방광암-기인성 사망 또는 진행(질병 재발 후 TNM 단계 진행으로 정의)의 가능성 등의 병의 경과 및 완치 여부를 의미한다. 본 발명의 목적상 예후는 방광암 치료 후 재발 가능성 또는 근침윤성 방광암(Muscle Invasive Bladder Cancer; MIBC)으로 진행 여부를 예측하는 것을 의미한다.As used herein, the term "prognosis" refers to bladder cancer-induced death or progression (disease recurrence), including recurrence (defined as recurrence of primary NMIBC of the same pathological stage), metastatic spread, and drug resistance of a disease such as bladder cancer. It means the course of the disease and whether it is cured or not, such as the possibility of post-TNM stage progression). For the purpose of the present invention, prognosis means predicting the possibility of recurrence or progress to muscle invasive bladder cancer (MIBC) after bladder cancer treatment.
본 발명에서 용어, "양호한 예후"는 암 환자, 특히 방광암 환자의 질병이 완치될 가능성을 의미하고, "양호하지 않은 예후"는 투병 중인 암 또는 종양의 진행(progression) 및 재발(recurrence), 전이 또는 사망할 가능성이 있음을 의미한다. 양호한 결과를 갖는 것으로 분류된 암 환자는 투병 중인 암 또는 종양이 없는 상태이다. 이와는 반대로, 양호하지 않은 결과의 암 환자는 질병의 재생, 종양 재발, 진행, 전이 또는 사망에 이른다. "양호한 예후"는 방광암 환자가 투병 중인 암 또는 종양이 적어도 2년, 보다 구체적으로는 적어도 5년 이상 동안 없는 상태로 있을 수 있음을 의미한다. 본 발명의 다른 측면에서, "양호하지 않은 예후"는 방광암 환자가 5년 미만 이내에 질병 재생, 종양 재발, 진행, 전이 또는 사망을 경험할 수 있음을 의미한다.In the present invention, the term "good prognosis" refers to the possibility of a cancer patient, particularly a bladder cancer patient, being cured of the disease, and "unfavorable prognosis" refers to the progression, recurrence, and metastasis of a cancer or tumor being treated. or likely to die. A cancer patient classified as having a good outcome is one who is fighting cancer or is free of a tumor. Conversely, poor outcome cancer patients undergo disease regeneration, tumor recurrence, progression, metastasis or death. "Good prognosis" means that a bladder cancer patient can remain free of the cancer or tumor he is fighting for at least 2 years, more specifically at least 5 years or more. In another aspect of the invention, "poor prognosis" means that a bladder cancer patient may experience disease regeneration, tumor recurrence, progression, metastasis, or death within less than 5 years.
본 발명에서 용어, "예측"이란 환자가 화학요법 또는 방사선 치료 등의 치료법에 대해 선호적으로 또는 비선호적으로 반응하여 환자가 치료, 예를 들어 특정 치료제, 및/또는 원발성 종양의 수술로 제거, 및/또는 암의 재발 없이 특정 시기 동안 화학요법으로 치료된 후의 생존 여부 및/또는 가능성과 관련된다. 본 발명의 예측방법은 임의의 특정 환자에 대한 가장 적절한 치료방식을 선택하여 적용함으로써 임상적으로 사용될 수 있다. 본 발명의 예측방법은 환자가, 예를 들어 소정의 치료제 또는 조합물, 외과적 개입, 화학요법 등의 투여를 비롯한 소정의 치료 처방과 같은 치료법에 선호적으로 반응하는지를 확인하거나, 치료 처방 후 환자의 장기 생존 또는 전신 또는 국소 재발이 가능한지를 예측할 수 있다. 또한 이를 통하여 불필요한 보조 항암요법을 최소화하거나 전신 또는 국소 재발이 예측되는 환자에게는 더욱 효과적인 보조 항암요법을 사용할 수 있도록 계획할 수 있다.As used herein, the term "prediction" means that a patient responds preferentially or non-preferably to a treatment such as chemotherapy or radiation treatment, so that the patient is treated, for example, a specific therapeutic agent, and / or surgical removal of a primary tumor, and/or survival and/or likelihood after being treated with chemotherapy for a specified period of time without recurrence of the cancer. The prediction method of the present invention can be used clinically by selecting and applying the most appropriate treatment method for any specific patient. The predictive method of the present invention confirms whether a patient responds preferentially to a treatment regimen, such as a predetermined treatment regimen, including, for example, the administration of a predetermined therapeutic agent or combination, surgical intervention, chemotherapy, or the like, or the patient after a treatment regimen of long-term survival or possible systemic or local recurrence. In addition, through this, it is possible to plan to minimize unnecessary adjuvant chemotherapy or to use more effective adjuvant chemotherapy for patients who are predicted to have systemic or local recurrence.
본 발명에서 용어, "예후 예측용 마커", "예후 예측을 위한 마커" 또는 "예후 예측 마커(prognosis marker)"란 방광암 세포를 정상 세포와 구분하여 방광암 치료 후 진행 또는 재발 여부를 비롯한 예후를 예측할 수 있는 물질이다. 본 발명의 목적상 비근침윤성 방광암 진단 후 수술 또는 화학요법을 시술받고 5년 이내에 근침윤성 방광암으로의 진행 여부 또는 재발 여부를 결정하여 예후가 양호한 환자와 양호하지 않은 환자를 구별할 수 있는 마커를 의미한다.In the present invention, the term "marker for predicting prognosis", "marker for predicting prognosis" or "prognosis marker" means to distinguish bladder cancer cells from normal cells and predict prognosis including progression or recurrence after bladder cancer treatment. It is a substance that can For the purpose of the present invention, it means a marker that can distinguish patients with good prognosis from patients with poor prognosis by determining whether or not to progress to or recur within 5 years after surgery or chemotherapy after diagnosis of non-muscle-invasive bladder cancer do.
유의성 있는 예후 예측 마커의 선택과 적용은 예후 예측 결과의 신뢰도를 결정짓는다. "유의성 있는 예후 예측 마커"란, 예후 예측하여 얻은 결과가 정확하여 타당도(validity)가 높고 반복 측정 시에도 일관된 결과를 나타내도록 신뢰도(reliability)가 높은 마커를 의미한다. 본 발명에 따른 방광암의 예후 예측 마커는, 비근침윤성 방광암 환자 조직을 이용한 RNA 시퀀싱 분석과 더불어 다양한 방광암 환자 코호트에 공통적으로 나타나는 차등적으로 발현된 유전자들로서, 반복된 실험에도 동일한 결과를 나타낸다.The selection and application of significant prognostic markers determines the reliability of prognostic prediction results. A "significant prognostic predictive marker" means a marker that has high validity because the result obtained by predicting prognosis is accurate and high reliability so as to show consistent results even during repeated measurements. The prognostic markers of bladder cancer according to the present invention are differentially expressed genes common to various bladder cancer patient cohorts in addition to RNA sequencing analysis using tissues of non-muscle invasive bladder cancer patients, and show the same results in repeated experiments.
본 발명에서 방광암의 예후 예측 마커로 선택된 상기 유전자들과 방광암의 진행 또는 재발과의 관련성에 대해서는 현재까지 보고된 바 없다.The relationship between the genes selected as prognostic markers for bladder cancer in the present invention and progression or recurrence of bladder cancer has not been reported to date.
또 하나의 양태로서, 본 발명은 BUB1 유전자의 mRNA 발현수준 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정하는 제제를 포함하는, 비근침윤성 방광암의 예후 예측용 조성물에 관한 것이다.In another aspect, the present invention relates to a composition for predicting the prognosis of non-muscle invasive bladder cancer, including an agent for measuring the mRNA expression level of the BUB1 gene or the expression level of a protein encoded by the gene.
본 발명의 일구체예에서, 상기 조성물은 UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 및 SPAG5로 이루어진 군으로부터 선택되는 하나 이상의 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정하는 제제를 더 포함할 수 있다.In one embodiment of the present invention, the composition is from the group consisting of UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 and SPAG5 It may further include an agent for measuring the expression level of mRNA of one or more selected genes or proteins encoded by the genes.
본 발명에서 용어, "mRNA 발현수준 측정"은 방광암의 예후를 예측하기 위하여 생물학적 시료에서 마커 유전자의 mRNA 존재 여부와 발현정도를 확인하는 과정으로, mRNA의 양을 측정함으로써 알 수 있다. 이를 위한 분석방법으로는 RT-PCR, 경쟁적 RT-PCR(competitive RT-PCR), 실시간 RT-PCR(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(northern blotting), DNA 마이크로어레이 칩 등이 있으나, 이들로 한정되는 것은 아니다.In the present invention, the term "mRNA expression level measurement" is a process of confirming the presence or absence of mRNA of a marker gene and the expression level in a biological sample in order to predict the prognosis of bladder cancer, and can be known by measuring the amount of mRNA. Analysis methods for this include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA; RNase protection assay), northern blotting blotting), DNA microarray chips, etc., but are not limited thereto.
본 발명에 따른 방광암의 예후 마커 유전자의 mRNA 수준을 측정하는 제제는 바람직하게는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브이며, 상기 마커 유전자의 염기서열을 바탕으로 이들 유전자의 특정 영역을 특이적으로 증폭하는 프라이머 또는 프로브를 고안할 수 있다. 본 발명에 따른 방광암의 예후 마커 유전자의 염기서열은 유전자뱅크(GenBank)에 등록되어 당해분야에 공지된 상태이므로, 당업자는 상기 염기서열을 바탕으로 이들 유전자의 특정 영역을 특이적으로 증폭할 수 있는 프라이머 또는 프로브를 디자인할 수 있다.The agent for measuring the mRNA level of a prognostic marker gene for bladder cancer according to the present invention is preferably an antisense oligonucleotide, a primer pair or a probe, which specifically amplifies a specific region of these genes based on the nucleotide sequence of the marker gene. Primers or probes can be designed. Since the nucleotide sequence of the prognostic marker gene for bladder cancer according to the present invention is registered in GenBank and is known in the art, those skilled in the art can specifically amplify a specific region of these genes based on the nucleotide sequence Primers or probes can be designed.
본 발명에서 용어, "안티센스"는 안티센스 올리고머가 왓슨-크릭 염기쌍 형성에 의해 RNA 내의 표적서열과 혼성화되어 표적 서열 내에서 전형적으로 mRNA와 헤테로이중체를 형성할 수 있는 뉴클레오티드 염기서열 및 서브 유닛간 백본을 갖는 올리고머를 지칭한다. 올리고머는 표적 서열에 대한 정확한 서열 상보성 또는 유사 상보성을 가질 수 있다. 이 안티센스 올리고머는 mRNA의 번역을 차단 또는 저해하고 mRNA의 스플라이스 변이체를 생산하는 mRNA의 프로세싱 과정을 변화시킬 수 있다.As used herein, the term "antisense" refers to a nucleotide sequence and a backbone between subunits in which an antisense oligomer can hybridize with a target sequence in RNA by Watson-Crick base pairing to form a heteroduplex with mRNA, typically in the target sequence. Refers to an oligomer having The oligomer may have exact sequence complementarity or near complementarity to the target sequence. These antisense oligomers can block or inhibit translation of mRNA and alter the processing of mRNA to produce splice variants of mRNA.
본 발명에서 용어, "프라이머"는 적절한 완충액 중의 적절한 조건(예를 들면, 4개의 다른 뉴클레오시드 트리포스페이트 및 DNA, RNA 폴리머라제 또는 역전사 효소와 같은 중합제) 및 적절한 온도 하에서 주형-지시 DNA 합성의 시작점으로서 작용할 수 있는 단일가닥 올리고뉴클레오티드를 말한다. 상기 프라이머의 적절한 길이는 사용 목적에 따라 달라질 수 있으나, 통상 15 내지 30개 뉴클레오티드이다. 짧은 프라이머 분자는 일반적으로 주형과 안정한 혼성체를 형성하기 위해서 더 낮은 온도를 필요로 한다. 프라이머 서열은 주형과 완전하게 상보적일 필요는 없으나, 주형과 혼성화할 정도로 충분히 상보적이어야 한다. 본 발명에서는 본 발명에 따른 방광암의 예후 마커 유전자에 대한 정방향 및 역방향 프라이머를 사용하여 PCR 증폭을 수행한 후 PCR 생성물의 증폭 여부를 통해 방광암의 예후를 예측할 수 있다.As used herein, the term "primer" refers to template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates and a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer and appropriate temperature. It refers to a single-stranded oligonucleotide that can serve as a starting point for The appropriate length of the primer may vary depending on the purpose of use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be perfectly complementary to the template, but must be sufficiently complementary to hybridize with the template. In the present invention, PCR amplification is performed using the forward and reverse primers for the prognostic marker gene for bladder cancer according to the present invention, and then the prognosis of bladder cancer can be predicted through whether or not the PCR product is amplified.
본 발명에서 용어, "프로브"는 mRNA와 특이적 결합을 이룰 수 있는 짧게는 수개 염기 내지 길게는 수백 개 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미한다. 프로브는 올리고 뉴클레오티드 프로브, 단일가닥 DNA 프로브, 이중가닥 DNA 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 본 발명에서는 본 발명에 따른 마커 유전자에 대해 상보적인 프로브를 이용하여 혼성화를 실시한 후, 혼성화 여부를 통해 방광암의 재발 여부를 비롯한 예후를 예측할 수 있다. 적당한 프로브의 선택 및 혼성화 조건은 당해분야에 공지된 것을 기초로 변형할 수 있다.In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundred bases as short as possible to achieve specific binding with mRNA. The probe may be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, or an RNA probe. In the present invention, after hybridization is performed using a probe complementary to the marker gene according to the present invention, prognosis including recurrence of bladder cancer can be predicted through hybridization. Selection of suitable probes and hybridization conditions can be modified based on those known in the art.
본 발명에 따른 안티센스 올리고뉴클레오티드, 프라이머 또는 프로브는 포스포르아미다이트 고체 지지체 방법을 비롯한 당해분야에 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, 캡핑, 천연 뉴클레오티드 하나 이상의 동족체로의 치환, 및 뉴클레오티드간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.Antisense oligonucleotides, primers or probes according to the present invention can be chemically synthesized using methods well known in the art, including the phosphoramidite solid support method. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more natural nucleotides with homologs, and modifications between nucleotides, such as uncharged linkages such as methylphosphonates, phosphotriesters, phosphoro amidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).
본 발명에서 용어, "단백질의 발현수준 측정"은 방광암의 진행 또는 재발 가능성을 예후 예측하기 위하여 생물학적 시료에서 마커 유전자로부터 코딩된 단백질의 존재 여부와 발현수준을 확인하는 과정으로, 상기 단백질에 대하여 특이적으로 결합하는 항체를 이용해 단백질의 양을 확인한다. 이를 위한 분석방법으로는 웨스턴 블랏팅(western blotting), ELISA(enzyme linked immunosorbent assay), 방사선면역분석법(radioimmunoassay), 방사면역확산법(radioimmunodiffusion), 오우크레로니(Ouchterlony) 면역확산법, 로케트(Rocket) 면역전기영동, 면역조직화학염색, 면역침전분석(immunoprecipitation assay), 보체고정분석(complete fixation assay), FACS, 단백질 칩(protein chip) 등이 있으나, 이들로 한정되는 것은 아니다.In the present invention, the term "measurement of protein expression level" is a process of confirming the presence and expression level of a protein encoded by a marker gene in a biological sample in order to predict the prognosis of the progression or recurrence of bladder cancer, and is specific for the protein. The amount of protein is confirmed using an antibody that binds antagonistically. Analysis methods for this include western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion method, Rocket immunoassay Electrophoresis, immunohistochemical staining, immunoprecipitation assay, complete fixation assay, FACS, protein chip, etc., but are not limited thereto.
본 발명에서 용어, "항체"는 당해분야에 공지된 용어로서, 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 본 발명의 마커 유전자로부터 코딩된 단백질에 대해 특이적으로 결합하는 항체를 의미하며, 이러한 항체는 각 유전자를 통상적인 방법에 따라 발현벡터에 클로닝하여 상기 마커 유전자에 의해 코딩되는 단백질을 얻은 후, 얻어진 단백질로부터 통상적인 방법에 의해 제조될 수 있다. 여기에는 상기 단백질에서 만들어질 수 있는 펩티드 단편도 포함되며, 본 발명의 펩티드 단편으로는, 최소한 7개 아미노산, 바람직하게는 9개 아미노산, 더욱 바람직하게는 12개 이상의 아미노산을 포함한다. 본 발명의 항체는 그 형태가 특별히 제한되지 않으며, 다중클론 항체, 단일클론 항체 또는 항원 결합성을 갖는 것이라면 그것의 일부도 본 발명의 항체에 포함되고, 모든 면역글로불린 항체가 포함된다.As used herein, the term "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to a protein encoded by a marker gene of the present invention, and such an antibody is obtained by cloning each gene into an expression vector according to a conventional method and using the marker gene. After obtaining the encoded protein, it can be produced from the obtained protein by conventional methods. This includes peptide fragments that can be made from the protein, and the peptide fragments of the present invention include at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids. The antibody of the present invention is not particularly limited in its form, and polyclonal antibodies, monoclonal antibodies, or any part thereof are included in the antibody of the present invention as long as they have antigen-binding properties, and all immunoglobulin antibodies are included.
상기한 바와 같이 방광암의 예후 예측 마커 유전자가 규명되었으므로, 이를 이용하여 항체를 생성하는 것은 당업계에 널리 공지된 기술을 이용하여 용이하게 제조할 수 있다. 다중클론 항체는 상기한 방광암의 예후 예측 마커 유전자로부터 코딩되는 단백질 항원을 동물에 주사하고 동물로부터 채혈하여 항체를 포함하는 혈청을 수득하는 당해분야에 널리 공지된 방법에 의해 생산할 수 있다. 이러한 다중클론 항체는 염소, 토끼, 양, 원숭이, 말, 돼지, 소 개 등의 임의의 동물 종 숙주로부터 제조 가능하다. 단클론 항체는 당해분야에 널리 공지된 하이브리도마 방법(hybridoma method)(Kohler 및 Milstein, European Jounral of Immunology, 6: 511-519, 1976), 또는 파지 항체 라이브러리(Clackson et al, Nature, 352: 624-628, 1991; Marks et al, J. Mol. Biol., 222(58): 1-597, 1991) 기술을 이용하여 제조될 수 있다. 상기 방법으로 제조된 항체는 겔 전기영동, 투석, 염침전, 이온교환 크로마토그래피, 친화성 크로마토그래피 등의 방법을 이용하여 분리, 정제할 수 있다.As described above, since the prognostic marker gene for bladder cancer has been identified, antibody production using the gene can be easily prepared using a technique well known in the art. The polyclonal antibody can be produced by a method well known in the art, in which a protein antigen encoded by the prognostic marker gene for bladder cancer is injected into an animal, blood is collected from the animal, and serum containing the antibody is obtained. Such polyclonal antibodies can be prepared from any animal species host, such as goat, rabbit, sheep, monkey, horse, pig, cow and the like. Monoclonal antibodies can be prepared by the hybridoma method well known in the art (Kohler and Milstein, European Journal of Immunology, 6: 511-519, 1976), or by a phage antibody library (Clackson et al, Nature, 352: 624 -628, 1991; Marks et al, J. Mol. Biol., 222(58): 1-597, 1991) technique. The antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.
나아가, 본 발명의 항체에는 인간화 항체 등의 재조합 항체도 포함된다. 본 발명에 사용되는 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란 적어도 항원 결합기능을 보유하고 있는 단편을 뜻하며, Fab, F(ab'), F(ab')2, Fv 등이 있다.Furthermore, the antibodies of the present invention include recombinant antibodies such as humanized antibodies. Antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, Fv, and the like.
또 하나의 양태로서, 본 발명은 상기 조성물을 포함하는 비근침윤성 방광암의 예후 예측용 키트에 관한 것이다.As another aspect, the present invention relates to a kit for predicting the prognosis of non-muscle invasive bladder cancer comprising the composition.
본 발명의 키트는 비근침윤성 방광암의 예후 예측 마커인 BUB1 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정(확인)함으로써 피험체를 대상으로 하여 비근침윤성 방광암의 예후를 예측하는데 사용될 수 있다. 본 발명의 비근침윤성 방광암의 예후 예측용 키트에는 상기 BUB1 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정하기 위한 폴리뉴클레오티드, 프라이머, 프로브 또는 항체뿐만 아니라 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물, 용액 또는 장치가 포함될 수 있다.The kit of the present invention can be used to predict the prognosis of non-muscle-invasive bladder cancer in a subject by measuring (confirming) the expression level of the mRNA of the BUB1 gene or the protein encoded by the gene, which is a prognostic marker of non-muscle-invasive bladder cancer. have. The kit for predicting the prognosis of non-muscle invasive bladder cancer of the present invention includes a polynucleotide, primer, probe or antibody for measuring the expression level of the mRNA of the BUB1 gene or the protein encoded by the gene, as well as one type or its suitable for the analysis method. Any of the above other component compositions, solutions or devices may be included.
구체적인 일례로서, 본 발명의 방광암의 예후 예측용 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. RT-PCR 키트는, 상기 BUB1 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드(dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNase, RNAse 억제제, DEPC-수(DEPC-water), 멸균수 등을 포함할 수 있다. 또한, 정량 대조구로 사용되는 유전자에 특이적인 프라이머 쌍을 포함할 수 있다.As a specific example, the kit for predicting the prognosis of bladder cancer of the present invention may be a kit including essential elements required to perform RT-PCR. The RT-PCR kit contains, in addition to each pair of primers specific for the BUB1 gene, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcription enzymes such as enzymes, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. In addition, a primer pair specific to a gene used as a quantitative control may be included.
다른 일례로서, 본 발명의 키트는 유전자 칩 분석법을 수행하기 위해 필요한 필수 요소를 포함할 수 있다. 유전자 칩 분석용 키트는, 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판, 및 형광표식 프로브를 제작하기 위한 시약, 제제, 효소 등을 포함할 수 있다. 또한, 기판은 정량 대조구 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있다.As another example, the kit of the present invention may include essential elements required to perform a gene chip assay. A gene chip analysis kit may include a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, reagents, enzymes, and the like for preparing a fluorescently labeled probe. In addition, the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.
본 발명의 키트는 상기 BUB1 유전자 이외에 UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 및 SPAG5로 이루어진 군으로부터 선택되는 하나 이상의 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정하는 제제를 더 포함할 수 있다.In addition to the BUB1 gene, the kit of the present invention is selected from the group consisting of UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 and SPAG5 It may further include an agent for measuring the expression level of mRNA of one or more genes or proteins encoded by the genes.
또 하나의 양태로서, 본 발명은 상기 조성물을 포함하는 비근침윤성 방광암의 예후 예측용 마이크로어레이에 관한 것이다.In another aspect, the present invention relates to a microarray for predicting the prognosis of non-muscle invasive bladder cancer comprising the above composition.
상기 마이크로어레이는 본 발명의 상기 선발된 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정하기 위한 폴리뉴클레오티드, 프라이머, 프로브 또는 항체를 포함하는 것을 제외하고는 통상적인 마이크로어레이로 이루어질 수 있다.The microarray may be composed of a conventional microarray except for including polynucleotides, primers, probes, or antibodies for measuring the expression level of the mRNA of the selected gene or the protein encoded by the gene of the present invention. have.
마이크로어레이 상에서의 핵산의 혼성화 및 혼성화 결과의 검출은 당업계에 잘 알려져 있다. 상기 검출은 예를 들면, 핵산 시료를 형광 물질, 예를 들면, Cy3 및 Cy5와 같은 물질을 포함하는 검출 가능한 신호를 발생시킬 수 있는 표지 물질로 표지한 다음, 마이크로어레이 상에 혼성화하고 상기 표지 물질로부터 발생하는 신호를 검출함으로써 혼성화 결과를 검출할 수 있다.Hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection is, for example, by labeling a nucleic acid sample with a fluorescent material, for example, a label material capable of generating a detectable signal including materials such as Cy3 and Cy5, followed by hybridization on a microarray and the labeling material. A hybridization result can be detected by detecting a signal arising from
또 다른 하나의 양태로서, 본 발명은 개체로부터 분리된 생물학적 시료에서 BUB1 유전자의 mRNA 발현수준 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정하는 단계를 포함하는, 비근침윤성 방광암의 예후 예측을 위한 정보를 제공하는 방법에 관한 것이다.In another aspect, the present invention is for predicting the prognosis of non-muscle invasive bladder cancer, comprising measuring the mRNA expression level of the BUB1 gene or the expression level of the protein encoded by the gene in a biological sample isolated from the subject. It is about how to present information.
본 발명의 일구체예에서, 상기 단계는 UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 및 SPAG5로 이루어진 군으로부터 선택되는 하나 이상의 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 더 측정할 수 있다.In one embodiment of the present invention, said step is from the group consisting of UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 and SPAG5 The expression level of the mRNA of one or more selected genes or the protein encoded by the gene can be further measured.
본 발명에서 용어, "개체로부터 분리된 생물학적 시료"란 방광암에 대한 수술 및/또는 화학요법을 시술 받은 개체로부터 분리된 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 또는 소변과 같은 시료 등을 포함하나, 이들로 한정되는 것은 아니다.As used herein, the term "biological sample isolated from an individual" refers to a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine isolated from an individual who has undergone surgery and/or chemotherapy for bladder cancer. Including, but not limited to, and the like.
mRNA 발현수준을 측정하기 위한 분석방법으로는 역전사효소 중합효소반응, 경쟁적 역전사효소 중합효소반응, 실시간 역전사효소 중합효소반응, RNase 보호 분석법, 노던 블랏팅, DNA 마이크로어레이 칩 등이 있으나, 이들로 한정되는 것은 아니다.Analysis methods for measuring mRNA expression levels include, but are limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting, and DNA microarray chip. it is not going to be
상기 분석방법을 통하여, 정상 대조군 시료의 mRNA 발현수준과 방광암 진행 또는 재발 의심 개체에서의 mRNA 발현수준을 비교할 수 있고, 마커 유전자에서 mRNA로의 발현수준의 유의한 증가여부를 판단하여 방광암 진행 또는 재발 의심 개체의 실제 방광암 진행 또는 재발 여부를 예후 예측할 수 있다. mRNA 발현수준은, 바람직하게는 마커로 사용되는 유전자에 특이적인 프라이머를 이용하는 역전사효소 중합반응 또는 마커로 사용되는 유전자에 특이적인 프로브를 이용하는 DNA 마이크로어레이 칩을 이용하여 측정할 수 있다.Through the above analysis method, it is possible to compare the mRNA expression level of a normal control sample and the mRNA expression level of an individual suspected of bladder cancer progression or recurrence, and determine whether or not a significant increase in the expression level from a marker gene to mRNA is suspected of bladder cancer progression or recurrence. It is possible to predict the actual progression or recurrence of bladder cancer in an individual. The mRNA expression level can be preferably measured using a reverse transcriptase polymerization reaction using primers specific to the gene used as a marker or a DNA microarray chip using a probe specific to the gene used as a marker.
본 발명의 바람직한 실시예에 따르면, 마커 유전자에 특이적인 프라이머를 사용하여 역전사효소 중합반응을 수행한 후 생성물을 전기영동하여 밴드 패턴과 밴드의 두께를 확인함으로써 방광암의 예후 예측 마커로 사용되는 유전자의 mRNA 발현수준을 측정한 후 이를 정상 대조군의 발현수준과 비교함으로써 방광암의 진행 또는 재발 가능성을 간편하게 예후 예측할 수 있다.According to a preferred embodiment of the present invention, reverse transcriptase polymerization is performed using primers specific to the marker gene, and the product is subjected to electrophoresis to confirm the band pattern and thickness of the band. After measuring the mRNA expression level, it is possible to easily predict the prognosis of the possibility of progression or recurrence of bladder cancer by comparing it with the expression level of a normal control group.
또한, 상기 마커 유전자에 의해 코딩되는 단백질 수준 측정은 항체를 이용할 수 있는데, 이러한 경우, 생물학적 시료 내의 상기 마커 유전자에 의해 코딩되는 단백질과 이에 특이적인 항체 결합물은, 즉, 항원-항체 복합체를 형성하며, 항원-항체 복합체의 형성량은 검출 라벨(detection label)의 시그널의 크기를 통해서 정량적으로 측정할 수 있다. 이러한 검출 라벨은 효소, 형광물, 리간드, 발광물, 미소입자(microparticle), 레독스 분자 및 방사선 동위원소로 이루어진 그룹 중에서 선택할 수 있으며, 이에 제한되는 것은 아니다. 단백질 수준을 측정하기 위한 분석 방법으로는, 이에 제한되지는 않으나, 웨스턴 블럿, ELISA, 방사선면역분석, 방사선 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 면역조직화학법, 면역침전분석법, 보체 고정분석법, FACS, 단백질 칩 등이 있다.In addition, an antibody can be used to measure the level of the protein encoded by the marker gene. In this case, the protein encoded by the marker gene in a biological sample and an antibody binding product specific thereto form an antigen-antibody complex. The formation amount of the antigen-antibody complex can be quantitatively measured through the size of the signal of the detection label. These detection labels may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes, but are not limited thereto. Assay methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunoassay, Oukteroni immunodiffusion assay, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay , complement fixation assay, FACS, and protein chip.
따라서 본 발명은 상기와 같은 검출 방법들을 통하여, 정상대조군 샘플에서의 마커 유전자의 mRNA 발현양 또는 단백질의 양과 비근침윤성 방광암으로 약물 치료 또는 종양 제거 수술을 받은 환자에서의 마커 유전자의 mRNA 발현양 또는 단백질의 양을 확인할 수 있고, 상기 발현양의 정도를 대조군과 비교함으로써 방광암의 진행 또는 재발을 예측할 수 있다.Therefore, the present invention, through the above detection methods, the amount of mRNA expression or protein of a marker gene in a normal control sample and the amount of mRNA expression or protein of a marker gene in a patient who has undergone drug treatment or tumor removal surgery for non-muscle invasive bladder cancer The amount of can be confirmed, and the progression or recurrence of bladder cancer can be predicted by comparing the level of expression with the control group.
본 발명에서는 비근침윤성 방광암으로 약물 치료 또는 종양 제거 수술을 받은 환자에서 상기 마커 유전자의 발현양 또는 단백질의 발현양이 정상대조군 시료에 비해 증가된 경우, 근침윤성 방광암으로 진행 가능성이 높거나 또는 비근침윤성 방광암이 재발할 가능성이 높을 것으로 예상할 수 있다.In the present invention, when the expression level of the marker gene or the expression level of the protein is increased compared to the normal control sample in patients who have undergone drug treatment or tumor removal surgery for non-muscle-invasive bladder cancer, the possibility of progressing to muscle-invasive bladder cancer is high or non-muscle-invasive bladder cancer Bladder cancer can be expected to be more likely to recur.
따라서 본 발명의 정보제공방법을 통해 비근침윤성 방광암으로 약물 치료 또는 종양 제거 수술을 받은 환자에서 근침윤성 방광암으로 진행 가능성이 높을 것으로 예상되거나, 또는, 비근침윤성 방광암이 재발할 가능성이 높을 것으로 예상된 환자군을 미리 선별할 수 있으므로, 보다 적극적인 치료가 가능하다.Therefore, a patient group who is expected to have a high possibility of progressing to muscle invasive bladder cancer or a high possibility of recurrence of non-muscle invasive bladder cancer in patients who have undergone drug treatment or tumor removal surgery for non-muscle invasive bladder cancer through the information providing method of the present invention can be selected in advance, so more aggressive treatment is possible.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.
<실시예><Example>
1. 연구 설계1. Study design
도 1은 본 발명의 전체적인 연구 설계를 나타낸 것이다. RNA-시퀀싱 분석은 정상대조군과 비교하여 비근침윤성 방광암(Non-Muscle Invasive Bladder Cancer; NMIBC) 환자에서 차등적으로 발현되는 후보 유전자를 스크리닝하기 위해 수행되었다. 배수 변화(fold change)> 2 및 P <0.01의 조건에서 270개의 상향 조절된 유전자와 908개의 하향 조절된 유전자가 확인되었다. 상기 데이터를 이전에 보고된 방광암 프로파일링 연구에서 확인된 969개의 유전자와 비교하였으며, 그 결과 방광암과 정상 조직 사이의 19개의 차등적으로 발현된 유전자(UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, BUB1, MCM2, RECQL4 및 SPAG5)를 최종적으로 규명하였다. 이렇게 선별된 19개 유전자의 생물학적 특징을 유전자 온톨로지(GO) 분석을 통해 상기 유전자가 핵 분열, 방추 조직 및 세포질 분열과 같은 세포주기의 중요한 단계에 관련되어 있음을 확인하였다(도 2 참조). 그 중 방광암의 신규 유전자 마커로 BUB1을 선택하여 추가 검증을 진행하였다. 트레이닝 코호트(training cohort)에서 비근침윤성 방광암(NMIBC) 환자(n = 86) 및 정상대조군(n = 15)에서 BUB1의 mRNA 발현은 실시간 PCR 분석을 통해 측정하였다. 마지막으로, 비근침윤성 방광암(NMIBC)에서 BUB1의 예후력을 내부 검증 코호트(GSE13507)와 외부 검증 코호트(E-MTAB-4321) 평가를 통해 검증하였다.Figure 1 shows the overall study design of the present invention. RNA-sequencing analysis was performed to screen for candidate genes that are differentially expressed in Non-Muscle Invasive Bladder Cancer (NMIBC) patients compared to normal controls. Under the condition of fold change > 2 and P <0.01, 270 up-regulated genes and 908 down-regulated genes were identified. The data were compared with 969 genes identified in previously reported bladder cancer profiling studies, and the results showed that 19 differentially expressed genes between bladder cancer and normal tissues (UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, BUB1, MCM2, RECQL4 and SPAG5) were finally identified. Through gene ontology (GO) analysis of the biological characteristics of the 19 genes thus selected, it was confirmed that the genes are involved in important stages of the cell cycle, such as nuclear division, spindle organization, and cytoplasmic division (see FIG. 2). Among them, BUB1 was selected as a novel genetic marker for bladder cancer and further verification was performed. The mRNA expression of BUB1 in non-muscle invasive bladder cancer (NMIBC) patients (n = 86) and normal controls (n = 15) in a training cohort was measured by real-time PCR analysis. Finally, the prognostic power of BUB1 in non-muscle invasive bladder cancer (NMIBC) was verified through evaluation of an internal validation cohort (GSE13507) and an external validation cohort (E-MTAB-4321).
2. 재료 및 방법2. Materials and Methods
방광암 환자 조직 임상 샘플Bladder cancer patient tissue clinical sample
연구 방법론은 헬싱키 선언에서 정한 표준을 준수하였다. 표 1은 RNA-시퀀싱 분석에 사용된 케이스 피험자의 임상 정보를 나타낸 것이다. 총 24개의 방광 조직 샘플을 분석에 사용하였다. 샘플은 16개의 비근침윤성 방광암(NMIBC) 환자 조직 및 방광암이 포함되지 않은 정상 주변 조직(Normal adjacent tissues; NAT) 8개로 구성되었다. The research methodology complied with the standards established by the Declaration of Helsinki. Table 1 shows the clinical information of case subjects used for RNA-sequencing analysis. A total of 24 bladder tissue samples were used for analysis. The samples consisted of 16 non-muscularly invasive bladder cancer (NMIBC) patient tissues and 8 normal adjacent tissues (NAT) that did not contain bladder cancer.
이후, 본 연구에서는 총 101개의 방광 조직을 실시간 PCR 분석에 사용했으며 임상 정보는 표 2에 자세히 나타내었다. 101개의 조직 샘플 중 86개는 원발성 비근침윤성 방광암(NMIBC) 환자에서 추출되었으며 조직학적으로 이행세포암종(transitional cell carcinoma)으로 확인되었다. 나머지 15개는 대조군으로 사용되었다(암이 의심되는 환자에게서 채취했지만 최종 진단은 스트레스성 요실금 또는 알수 없는 혈뇨와 같은 양성 질환이었다). 연구에 사용된 생체 표본은 국립 바이오 뱅크 회원사인 충북대학교 병원(대한민국, 청주)에서 제공받았다. 이 연구는 충북대학교 기관 심의위원회(GR2010-12-010) 및 생명윤리위원회(IRB No 2020-07-018)의 승인을 받았으며 실험은 모든 참가자의 사전 서면 동의를 받아 수행되었다. 분석에 영향을 미치는 교란 요인의 가능성을 줄이기 위해 수반되는 제자리암(concomitant carcinoma in situ) 또는 상황 병변에만 있는 암종(carcinoma in situ lesions alone)으로 진단된 환자는 제외하였다. 2000년 4월부터 2010년 10월까지 충북대학교 병원에서 이행상피암종(transitional cell carcinoma)의 외과적 절제 과정에서 신선한 조직을 채취하였다. 모든 종양은 일반적으로 외과적 절제 후 15분 이내에 거시절제되었다. 각 샘플은 근치 방광 절제술 및 방광 종양의 경요도 절제술(Trans Urethral Resection of Bladder Tumor; TURBT)에서 얻은 신선한 냉동 시료의 일부에 대한 병리학적 분석을 통해 확인되었다. 종양은 표준 기준에 따라 단계(2002 TNM 분류) 및 등급(2004 WHO 분류)으로 분류되었다. 각 환자를 추적하고 표준 지침 권고에 따라 제안된 관리를 진행하였다. 유럽 비뇨기과 협회 지침에 따라 방광경 검사와 상부 요로 영상으로 관찰을 수행하였다. 재발은 동일한 병리학적 단계의 원발성 비근침윤성 방광암(NMIBC)의 재발로 정의되었으며, 비근침윤성 방광암(NMIBC)의 진행은 질병 재발 후 TNM 단계의 진행으로 정의되었다. 비근침윤성 방광암(NMIBC) 환자의 평균 추적 기간은 71.15 개월(범위, 10.70-174.90)이었다.Subsequently, in this study, a total of 101 bladder tissues were used for real-time PCR analysis, and the clinical information is detailed in Table 2. Of the 101 tissue samples, 86 were derived from patients with primary non-muscle invasive bladder cancer (NMIBC) and histologically confirmed as transitional cell carcinoma. The remaining 15 were used as controls (taken from patients with suspected cancer, but the final diagnosis was a benign disease such as stress incontinence or unknown hematuria). Biospecimens used in the study were provided by Chungbuk National University Hospital (Cheongju, Korea), a member company of the National Biobank. This study was approved by the Chungbuk National University Institutional Review Board (GR2010-12-010) and Bioethics Committee (IRB No 2020-07-018), and the experiment was conducted with prior written consent from all participants. To reduce the possibility of confounding factors affecting the analysis, patients diagnosed with concomitant carcinoma in situ or carcinoma in situ lesions alone were excluded. Fresh tissues were collected during surgical resection of transitional cell carcinoma at Chungbuk National University Hospital from April 2000 to October 2010. All tumors were macroscopically resected generally within 15 minutes of surgical excision. Each sample was identified by pathological analysis of a portion of a fresh frozen sample obtained from radical cystectomy and Trans Urethral Resection of Bladder Tumor (TURBT). Tumors were classified into stage (2002 TNM classification) and grade (2004 WHO classification) according to standard criteria. Each patient was followed up and the proposed management proceeded according to standard guidelines recommendations. Observations were performed with cystoscopy and upper urinary tract imaging according to the guidelines of the European Urological Association. Recurrence was defined as recurrence of primary non-muscle-invasive bladder cancer (NMIBC) of the same pathological stage, and progression of non-muscle-invasive bladder cancer (NMIBC) was defined as progression to the TNM stage after disease recurrence. The mean follow-up period for patients with non-muscle invasive bladder cancer (NMIBC) was 71.15 months (range, 10.70-174.90).
NMIBC, non-muscle invasive bladder cancer; SD, standard deviation.NMIBC, non-muscle invasive bladder cancer; SD, standard deviation.
* t-검정(student t-test)를 통해 얻은 P 값 * P value obtained through student t-test
# 카이제곱 검정(Chi-squared test)을 통해 얻은 P 값 # P value obtained through Chi-squared test
BCG, Bacillus Calmette-Guerin; NMIBC, non-muscle invasive bladder cancer; SD, standard deviation.BCG, Bacillus Calmette-Guerin; NMIBC, non-muscle invasive bladder cancer; SD, standard deviation.
* t-검정(student t-test)를 통해 얻은 P 값 * P value obtained through student t-test
# 카이제곱 검정(Chi-squared test)을 통해 얻은 P 값 # P value obtained through Chi-squared test
공개 발현 프로파일public expression profile
GSE13507(CBNUH microarray cohort)의 발현 프로파일은 GEO 온라인 데이터베이스(https://www.ncbi.nlm.nih.gov/geo/)에서 다운로드하였으며, E-MTAB-4321(UROMOL cohort)은 EMBL-EBI(http://www.ebi.ac.uk/)에서 다운로드하였다.The expression profile of GSE13507 (CBNUH microarray cohort) was downloaded from the GEO online database (https://www.ncbi.nlm.nih.gov/geo/), and E-MTAB-4321 (UROMOL cohort) was obtained from EMBL-EBI (http://www.ncbi.nlm.nih.gov/geo/). http://www.ebi.ac.uk/).
방광암 세포주bladder cancer cell line
인간 방광암 세포주인 5637 및 T24는 한국 세포주 은행(KCLB, 서울, 대한민국)에서 수득하였다. 두 세포주는 10% 소태아혈청(FBS)(Gibco, Thermo Fisher Scientific, Waltham, MA, USA) 및 1% 페니실린-스트렙토마이신(Gibco, Thermo Fisher Scientific, Waltham, MA, USA)이 보충된 RPMI 1640(Gibco)에서 5% CO2 인큐베이터에서 37℃ 조건 하에 배양되었다.Human bladder
RNA 시퀀싱 분석RNA sequencing analysis
시퀀싱 실험의 FASTQ 파일은 품질을 위해 처리되었으며 Trim Galore는 어댑터 시퀀스를 제거하는데 사용되었다. 처리된 raw read는 STAR를 사용하여 인간 게놈(hg38)에 정렬되었고 정렬된 읽기는 HOMER을 사용하여 태그 디렉토리로 변환되었다. 각 파일은 HOMER의 "analyzeRepeats"스크립트를 사용하여 정량화되었다. 각 샘플에서 유전자의 발현 수준의 정규화는 백만 개의 매핑된 판독(FPKM) 당 전사물의 킬로베이스 당 단편에 의해 이용되었다. 그런 다음, 조정된 P-value < 0.05 및 배수 변화(fold change) > 2 조건에서 HOMER의 "getDifferentialExpression"명령을 통해 DESeq2 분석에 의해 원시 읽기 카운트를 사용하여 차등적으로 발현된 유전자(DEGs)를 식별하였다. Metascape를 사용하여 유전자 온톨로지(GO) 분석을 수행하였다. 유전자세트증폭분석(gene set enrichment analysis, GSEA)을 수행하여 MSigDB에서 추출한 유전자 목록에서 증폭된 차등적으로 발현된 유전자(DEG)를 확인하여 Hallmark 및 큐레이트(C2) 컬렉션에서 유전자 세트의 증폭을 결정하였다.FASTQ files of sequencing experiments were processed for quality and Trim Galore was used to remove adapter sequences. The processed raw reads were aligned to the human genome (hg38) using STAR and the aligned reads were converted to a tag directory using HOMER. Each file was quantified using HOMER's "analyzeRepeats" script. Normalization of expression levels of genes in each sample was used by fragments per kilobase of transcript per million mapped reads (FPKM). Then, differentially expressed genes (DEGs) were identified using raw read counts by DESeq2 analysis via the "getDifferentialExpression" command in HOMER under conditions of adjusted P-value < 0.05 and fold change > 2 did Gene Ontology (GO) analysis was performed using Metascape. Perform gene set enrichment analysis (GSEA) to identify amplified differentially expressed genes (DEGs) from gene lists extracted from MSigDB to determine enrichment of gene sets from Hallmark and Curate (C2) collections. did
RT-qPCR analysisRT-qPCR analysis
[환자 조직][Patient organization]
전체 RNA는 이전에 설명한대로 TRIzol 시약(Invitrogen, Carlsbad, CA, USA)을 사용하여 조직에서 추출하고 -80℃에 보관하였다(Kim, W., et al., S100A9 and EGFR gene signatures predict disease progression in muscle invasive bladder cancer patients after chemotherapy. Annals of oncology, 2014. 25(5): p. 974-979.). 다음으로, 제조업체의 프로토콜에 따라 First Strand cDNA Synthesis Kit(Clontech, TAKARA, Otsu, Japan)를 사용하여 1ug의 전체 RNA에서 cDNA를 합성하였다. Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as previously described and stored at -80 °C (Kim, W., et al., S100A9 and EGFR gene signatures predict disease progression in muscle invasive bladder cancer patients after chemotherapy.Annals of oncology, 2014. 25(5): p. 974-979.). Next, cDNA was synthesized from 1 ug of total RNA using the First Strand cDNA Synthesis Kit (Clontech, TAKARA, Otsu, Japan) according to the manufacturer's protocol.
Rotor Gene 6000 기기(Qiagen, Hilden, Germany)를 사용하여 real-time PCR로 조직 mRNA를 증폭하고 2-ΔΔCt 방법을 사용하여 정량화하였다. Real-Time PCR은 제조사의 프로토콜에 따라 SYBR Premix Ex Taq Ⅱ(Clontech, TAKARA, Otsu, Japan)를 사용하여 수행되었다. PCR 반응은 5μL의 2x SYBR Premix EX Taq 버퍼, 0.5μL의 각 5 '및 3'프라이머(10pM/μL) 및 2μL의 샘플 cDNA를 포함하는 10μL의 최종 부피에서 수행되었다. 그 후 알려진 PCR 산물 농도를 100 pg/μL에서 0.1 pg/μL로 연속 10배 희석하여 표준 곡선을 설정하였다. Real-Time PCR 조건은 다음과 같다: 96℃에서 20초간 1 사이클 진행한 후, 이어서 변성(denaturation) 과정을 96℃에서 3초, 어닐링(annealing) 과정을 60℃에서 15초, 연장(extension) 과정을 72℃에서 15초간 40 사이클 진행하였고, melting program은 45 초당 1℃의 가열 속도로 72-95℃에서 수행되었다. Rotor-Gene Q software 2.3.1.49는 spectral data를 캡처하고 분석하는데 사용되었다. 모든 샘플은 triplicate로 실행되었다. 각 샘플에서의 유전자 발현값은 GAPDH의 발현값으로 정규화되었다. 다음 프라이머를 사용하여 후보 유전자를 증폭하였다. BUB1(Gene ID: 699), sense, 5'-AGCCCAGACAGTAACAGACTC-3 '(21bp, Tm 64℃); antisense, 5'-GTTGGCAACCTT ATGTGTTTCAC-3 '(23bp, Tm 66℃) 및 amplicon 크기는 113bp이다. 대조군 GAPDH(Gene ID: 2597) 프라이머는 다음과 같다: sense, 5'-CATGTTCGTCATGGGTGTGA-3 '(20bp, Tm 60℃); antisense, 5'-ATGGCATGGACTGTGGTCAT-3 '(20bp, Tm 60℃) 및 amplicon 크기는 156bp이다.Tissue mRNA was amplified by real-time PCR using a Rotor Gene 6000 machine (Qiagen, Hilden, Germany) and quantified using the 2-ΔΔCt method. Real-time PCR was performed using SYBR Premix Ex Taq II (Clontech, TAKARA, Otsu, Japan) according to the manufacturer's protocol. PCR reactions were performed in a final volume of 10 μL containing 5 μL of 2x SYBR Premix EX Taq buffer, 0.5 μL of each 5′ and 3′ primer (10 pM/μL) and 2 μL of sample cDNA. A standard curve was then established by serial 10-fold dilution of known PCR product concentrations from 100 pg/μL to 0.1 pg/μL. Real-Time PCR conditions are as follows: 1 cycle at 96 ° C for 20 seconds, followed by denaturation at 96 ° C for 3 seconds, annealing at 60 ° C for 15 seconds, extension The process was carried out 40 cycles at 72 ° C for 15 seconds, and the melting program was performed at 72-95 ° C at a heating rate of 1 ° C per 45 seconds. Rotor-Gene Q software 2.3.1.49 was used to capture and analyze the spectral data. All samples were run in triplicate. Gene expression values in each sample were normalized to that of GAPDH. Candidate genes were amplified using the following primers. BUB1 (Gene ID: 699), sense, 5′-AGCCCAGACAGTAACAGACTC-3′ (21 bp, Tm 64° C.); antisense, 5′-GTTGGCAACCTT ATGTGTTTCAC-3′ (23 bp, Tm 66 °C) and the amplicon size is 113 bp. Control GAPDH (Gene ID: 2597) primers were as follows: sense, 5′-CATGTTCGTCATGGGTGTGA-3′ (20bp,
[siRNA 형질주입 세포주][siRNA transfected cell line]
Ribospin II(GeneAll, Seoul, Korea)를 사용하여 세포에서 전체 RNA를 추출한 후 RevertAid First Strand cDNA Synthesis kit(Thermo Fisher Scientific, Waltham, MA, USA)를 사용하여 500ng의 전체 RNA를 역전사하였다.After total RNA was extracted from cells using Ribospin II (GeneAll, Seoul, Korea), 500 ng of total RNA was reverse transcribed using RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).
Real-time PCR은 TOPreal qPCR 2X PreMIX (SYBR Green with low ROX)(Enzynomics, Daejeon, Korea)를 사용하여 반복적으로 평가되었다. Rotor-Gene Q software 2.3.1은 spectral data를 캡처하고 분석하는데 사용되었다. 다음 프라이머를 사용하여 후보 유전자를 증폭하였다. BUB1(Gene ID : 699), sense, 5'-AGCCCAGACAGTAACAGACTC-3 '(21bp, Tm 60.9℃); antisense, 5'-GTTGGCAACCTTATGTGTTTCAC-3 '(23bp, Tm 60.5℃) 및 amplicon 크기는 136bp이다. 대조군 TBP(Gene ID : 6908) 프라이머는 다음과 같았다 : sense, 5'-CCCGAAACGCCGAATATAATCC-3 '(22bp, Tm 61.2℃); antisense, 5'-AATCAGTGCCGTGGTTCGTG-3 '(20bp, Tm 63℃) 및 amplicon 크기는 80bp이다.Real-time PCR was evaluated repeatedly using TOPreal qPCR 2X PreMIX (SYBR Green with low ROX) (Enzynomics, Daejeon, Korea). Rotor-Gene Q software 2.3.1 was used to capture and analyze the spectral data. Candidate genes were amplified using the following primers. BUB1 (Gene ID: 699), sense, 5′-AGCCCAGACAGTAACAGACTC-3′ (21 bp, Tm 60.9° C.); antisense, 5′-GTTGGCAACCTTATGTGTTTCAC-3′ (23 bp, Tm 60.5 °C) and the amplicon size is 136 bp. Control TBP (Gene ID: 6908) primers were as follows: sense, 5′-CCCGAAACGCCGAATATAATCC-3′ (22bp, Tm 61.2° C.); antisense, 5′-AATCAGTGCCGTGGTTCGTG-3′ (20 bp, Tm 63 °C) and the amplicon size is 80 bp.
siRNA 형질주입siRNA transfection
인간 BUB1 유전자 넉다운(knockdown)은 SMART pool ON-TARGETplus siRNA(Dharmacon : L-004102-00-0005)를 사용하였다. ON-TARGETplus Non-targeting Pool(Dharmacon: D-001810-10-05)을 대조군으로 사용하였다. 5637 및 T24 세포를 6 웰 플레이트에 분주하고 24시간 동안 배양한 다음, 항생제 없이 제조사의 프로코콜에 따라 Opti-MEM I reduced serum medium(Thermo Fisher Scientific, Waltham, MA, USA)에서 RNA 듀플렉스 및 Lipofectamine RNAi-MAX(Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA)로 형질주입하였다. 배지는 24시간 후에 항생제가 있는 완전배지로 변경하였으며, 24시간 후에 추가실험을 위해 세포를 수거하였다.For human BUB1 gene knockdown, SMART pool ON-TARGETplus siRNA (Dharmacon: L-004102-00-0005) was used. ON-TARGETplus Non-targeting Pool (Dharmacon: D-001810-10-05) was used as a control. 5637 and T24 cells were seeded in 6-well plates and cultured for 24 hours, followed by RNA duplex and Lipofectamine RNAi in Opti-MEM I reduced serum medium (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol without antibiotics. -MAX (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was transfected. The medium was changed to complete medium with antibiotics after 24 hours, and cells were harvested for further experiments after 24 hours.
유세포 분석flow cytometry
5637 및 T24 세포주를 비-표적 대조군 siRNA(siNC) 및 BUB1 siRNA(siBUB1)로 형질감염시켰다. 48시간 후, 세포를 수확하였다. 세포주기의 검출을 위해 세포를 PBS로 차가운 70% 에탄올에 고정시켰다. 이어서 세포주기 분석을 위해 프로피디움 요오드화물(Propidium Iodide, PI) 용액으로 세포를 염색하였다(Invitrogen: F10797). 세포의 DNA 함량은 CytoFLEX(Beckman Coulter, Brea, CA, USA)를 사용하여 유세포 분석(flow cytometry)에 의해 분석되었다. 데이터는 FlowJo software (TreeStar)를 사용하여 플로팅되었다.5637 and T24 cell lines were transfected with non-target control siRNA (siNC) and BUB1 siRNA (siBUB1). After 48 hours, cells were harvested. For cell cycle detection, cells were fixed in cold 70% ethanol with PBS. Subsequently, cells were stained with a propidium iodide (PI) solution for cell cycle analysis (Invitrogen: F10797). Cellular DNA content was analyzed by flow cytometry using CytoFLEX (Beckman Coulter, Brea, CA, USA). Data were plotted using FlowJo software (TreeStar).
통계 분석statistical analysis
연속 변수는 평균±표준편차(SD)로 표현되었다. 데이터의 정규성은 One-Sample Kolmogorov-Smirnov 검정으로 추정되었다. Welch의 t-검정은 두 개의 비 대응 샘플의 통계 분석에 사용되었으며 쌍을 이루는 t-검정은 쌍을 이루는 샘플의 통계 분석에 사용되었다. 오차 막대는 평균의 표준 오차(SEM)를 나타낸다. 그룹은 카이제곱 검정(Chi-squared test)에 의해 비 연속 변수 측면에서 비교되었다. 유전자 발현 값은 샘플 전체에서 자연 로그 변환 및 중앙값 중심이었다. Mann-Whitney U 테스트를 사용하여 실시간 PCR에 의해 NMIBC 조직 대 대조군 조직에서 BUB1의 발현을 조사하였다. P 값 <0.05는 유의성이 있는 것으로 간주되었다. 다양한 임상 병리학적 변수의 중요성은 단변량 및 다변량 Cox 비례 위험 회귀 모델을 사용하여 평가되었다. 상대 위험은 위험비(Hazard ratio, HR)와 95% 신뢰 구간(confidence intervals, CI)에 의해 결정되었다. Kaplan-Meier 생존 곡선을 그려 유전적 바이오 마커의 예후값을 결정하고 로그 순위 테스트를 사용하여 비교하였다. 통계 분석은 GraphPad Prism 8(GraphPad Software, San Diego, CA, USA) 및 IBM SPSS Statistics ver. 24.0(IBM Co., Armonk, NY, USA)을 이용하여 수행되었다. P 값 <0.05는 유의성이 있는 것으로 간주되었다. Continuous variables were expressed as mean ± standard deviation (SD). Normality of data was estimated with the One-Sample Kolmogorov-Smirnov test. Welch's t-test was used for statistical analysis of two unpaired samples and paired t-test was used for statistical analysis of paired samples. Error bars represent standard error of the mean (SEM). Groups were compared in terms of non-continuous variables by Chi-squared test. Gene expression values were natural log transformed and median centered across samples. Expression of BUB1 was examined in NMIBC tissues versus control tissues by real-time PCR using the Mann-Whitney U test. A P value <0.05 was considered significant. The significance of various clinicopathological variables was assessed using univariate and multivariate Cox proportional hazards regression models. Relative risk was determined by hazard ratio (HR) and 95% confidence intervals (CI). Kaplan-Meier survival curves were drawn to determine the prognostic value of genetic biomarkers and compared using the log-rank test. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) and IBM SPSS Statistics ver. 24.0 (IBM Co., Armonk, NY, USA). A P value <0.05 was considered significant.
2. 결과2. Results
스크리닝 테스트screening test
[비근침윤성 방광암(NMIBC) 환자의 전사체 프로파일][Transcriptomic Profile of Patients with Non-Muscularly Invasive Bladder Cancer (NMIBC)]
비근침윤성 방광암(NMIBC)에서 새로운 후보 예측 마커 유전자를 확인하기 위해 비근침윤성 방광암(NMIBC) 환자의 방광 종양(n=16) 및 정상 주변 조직(NAT; n=8)에서 RNA 시퀀싱을 수행하여 비근침윤성 방광암(NMIBC)의 유전자 발현 패턴을 확인하였다.To identify novel candidate predictive marker genes in non-muscle-invasive bladder cancer (NMIBC), RNA sequencing was performed in bladder tumors (n=16) and normal surrounding tissues (NAT; n=8) of patients with non-muscle-invasive bladder cancer (NMIBC). Gene expression patterns of bladder cancer (NMIBC) were identified.
주성분 분석(Principal component analysis; PCA)은 비근침윤성 방광암(NMIBC) 환자로부터 정상 주변 조직(NAT)과 종양의 전사체 프로파일을 분리하였다(도 3a 참조). 본 실험에서 정상 주변 조직(NAT)과 비교하여 비근침윤성 방광암(NMIBC)에서 1178개의 차등적으로 발현된 유전자(270개의 상향 조절된 유전자 및 908개의 하향 조절된 유전자)(FDR <0.05, > 2 fold differences in expression)를 확인하였다(도 3b 참조). 이중 비근침윤성 방광암(NMIBC)에서 크게 변경된 대표적인 유전자를 Volcano 플롯 그래프로 시각화하였다(도 3c 참조). 유전자 온톨로지(GO) 분석은 차등적으로 발현된 유전자의 기능성 프로파일을 밝혀냈다. 정상 주변 조직(NAT)에 비해 비근침윤성 방광암(NMIBC)에서 상향 조절된 유전자는 세포주기, DNA 패키징, 세포 분열, G2/M 체크 포인트 및 DNA 복구와 같은 경로(pathway)와 강하게 연관되어 있는 것으로 나타났으며; 반면에, 정상 주변 조직(NAT)에 비해 비근침윤성 방광암(NMIBC)에서 하향 조절된 유전자는 세포 외 기질 조직, 근육 시스템 과정 및 근육 구조 발달과 연관되어 있는 것으로 나타났다(도 3d 참조). 이러한 과정은 종양 발달 및 진행과 관련된 방광암의 잘 알려진 변화이다. 더욱이 유전자세트증폭분석(GSEA)은 또한 비근침윤성 방광암(NMIBC) 환자에서 상향 조절된 유전자가 세포주기 및 G2/M 체크 포인트 과정과 연관되어 있음을 보여주었다(도 3e 참조).Principal component analysis (PCA) separated normal peripheral tissue (NAT) and tumor transcriptome profiles from non-muscularly invasive bladder cancer (NMIBC) patients (see Fig. 3a). In this experiment, 1178 differentially expressed genes (270 upregulated genes and 908 downregulated genes) in non-muscle invasive bladder cancer (NMIBC) compared to normal surrounding tissue (NAT) (FDR < 0.05, > 2 fold) differences in expression) were confirmed (see Fig. 3b). Representative genes significantly altered in double non-muscle invasive bladder cancer (NMIBC) were visualized as Volcano plot graphs (see Fig. 3c). Gene Ontology (GO) analysis revealed functional profiles of differentially expressed genes. Genes upregulated in non-muscle invasive bladder cancer (NMIBC) compared to normal surrounding tissue (NAT) have been shown to be strongly associated with pathways such as cell cycle, DNA packaging, cell division, G2/M checkpoint and DNA repair. born; On the other hand, genes downregulated in non-muscle invasive bladder cancer (NMIBC) compared to normal surrounding tissue (NAT) were found to be associated with extracellular matrix organization, muscle system processes and muscle structure development (see Fig. 3d). This process is a well-known change in bladder cancer associated with tumor development and progression. Moreover, gene set amplification analysis (GSEA) also showed that genes upregulated in non-muscle invasive bladder cancer (NMIBC) patients were associated with cell cycle and G2/M checkpoint processes (see Fig. 3e).
상기 결과를 종합한 결과, 세포주기를 조절하는 유전자가 비근침윤성 방광암(NMIBC)에서 중요한 역할을 한다는 것을 알 수 있었다.As a result of the above results, it was found that genes regulating the cell cycle play an important role in non-muscle invasive bladder cancer (NMIBC).
[두 개의 다른 코호트에서 차등적으로 발현된 유전자 비교][Comparison of differentially expressed genes in two different cohorts]
RNA-시퀀싱 분석 결과 정상 주변 조직(NAT) 대비 비근침윤성 방광암(NMIBC) 환자에서 상향 조절된 270개의 유전자, GSE13507 데이터 세트에서 정상 대조군 대비 비근침윤성 방광암(NMIBC) 환자에서 상향 조절된 481개의 유전자, GSE13507 데이터 세트에서 비근침윤성 방광암(NMIBC) 대비 근침윤성 방광암(MIBC) 환자에서 상향 조절된 527개의 유전자를 교차시켜 비근침윤성 방광암(NMIBC) 예후를 위한 표적 이상치 유전자를 얻었다(도 1 다이아그램 참조). 따라서, 19개의 상향 조절된 유전자가 세 가지 범주 사이에 중첩되어 이러한 유전자가 방광암에 대한 가장 유망한 후보임을 확인하였다. 이렇게 선별된 19개 유전자의 생물학적 특징을 유전자 온톨로지(GO) 분석을 통해 상기 유전자가 핵 분열, 방추 조직 및 세포질 분열과 같은 세포주기의 중요한 단계에 관련되어 있음을 확인하였다(도 2 참조). 이 중 스핀들 체크 포인트 조절자(spindle checkpoint regulator)인 BUB1을 NMIBC 병인 및 예후에 미치는 역할을 평가하기 위해 최종적인 유전자로 선택하였다. RNA-시퀀싱 분석에서 비근침윤성 방광암(NMIBC) 및 정상 주변 조직(NAT)에서 BUB1의 발현은 도 4에 자세히 나타내었다.RNA-sequencing analysis results: 270 genes up-regulated in non-muscle-invasive bladder cancer (NMIBC) patients compared to normal peripheral tissue (NAT), GSE13507 481 genes up-regulated in non-muscle-invasive bladder cancer (NMIBC) patients compared to normal controls in data set, GSE13507 In the data set, 527 genes upregulated in non-muscle-invasive bladder cancer (NMIBC) versus non-muscle-invasive bladder cancer (MIBC) patients were crossed to obtain target outlier genes for non-muscle-invasive bladder cancer (NMIBC) prognosis (see Figure 1 diagram). Thus, 19 upregulated genes overlapped between the three categories, making these genes the most promising candidates for bladder cancer. Through gene ontology (GO) analysis of the biological characteristics of the 19 genes thus selected, it was confirmed that the genes are involved in important stages of the cell cycle, such as nuclear division, spindle organization, and cytoplasmic division (see FIG. 2). Among them, BUB1, a spindle checkpoint regulator, was selected as the final gene to evaluate its role in NMIBC pathogenesis and prognosis. The expression of BUB1 in non-muscle invasive bladder cancer (NMIBC) and normal peripheral tissue (NAT) in RNA-sequencing analysis is detailed in FIG. 4 .
트레이닝 테스트training test
[비근침윤성 방광암(NMIBC) 조직에서 BUB1 mRNA 발현][BUB1 mRNA expression in non-muscle invasive bladder cancer (NMIBC) tissue]
비근침윤성 방광암(NMIBC) 조직에서 BUB1의 mRNA 발현을 확인하기 위해 RT-qPCR 분석을 수행한 결과, BUB1이 대조군보다 비근침윤성 방광암(NMIBC) 조직에서 상당히 과발현되었음을 확인하였다(정상 방광 점막; P <0.0001)(도 5a 참조). 그리고 BUB1 mRNA는 비근침윤성 방광암(NMIBC)의 등급(grade)과 예후 변화에 따라 다르게 발현되는 것을 확인하였다. 고등급(High grade)의 비근침윤성 방광암(NMIBC) 환자에서 BUB1 발현은 저등급(low grade)의 비근침윤성 방광암(NMIBC) 환자에서의 발현보다 유의하게 높게 나타났다(P <0.01; 도 5b 참조). 더욱이, BUB1의 mRNA 발현은 진행이 없는 비근침윤성 방광암(NMIBC) 환자와 비교했을 때 나중에 근침윤성 방광암(MIBC)로 진행한 비근침윤성 방광암(NMIBC) 환자에서 더 높은 것으로 나타났다(P <0.05; 도 5c 참조). As a result of performing RT-qPCR analysis to confirm the mRNA expression of BUB1 in non-muscle-invasive bladder cancer (NMIBC) tissues, it was confirmed that BUB1 was significantly overexpressed in non-muscle-invasive bladder cancer (NMIBC) tissues than in the control group (normal bladder mucosa; P <0.0001). ) (see Fig. 5a). In addition, it was confirmed that BUB1 mRNA was expressed differently depending on the grade and prognosis of non-muscle invasive bladder cancer (NMIBC). The expression of BUB1 in patients with high-grade non-muscle-invasive bladder cancer (NMIBC) was significantly higher than that in patients with low-grade non-muscle-invasive bladder cancer (NMIBC) (P <0.01; see Fig. 5b). Moreover, the mRNA expression of BUB1 was found to be higher in non-muscle-invasive bladder cancer (NMIBC) patients who later progressed to MIBC compared to NMIBC patients without progression (P < 0.05; Fig. 5c). Reference).
이러한 결과는 비근침윤성 방광암(NMIBC)의 예후 지표로서 BUB1의 가능성을 보여주는 것이다.These results show the potential of BUB1 as a prognostic marker for non-muscle invasive bladder cancer (NMIBC).
[BUB1 mRNA 발현의 비근침윤성 방광암(NMIBC) 예후와의 관련성][Relationship of BUB1 mRNA expression with prognosis of non-muscle invasive bladder cancer (NMIBC)]
비근침윤성 방광암(NMIBC)에서 BUB1의 예후력을 평가하기 위해 생존 분석을 수행하였다. 비근침윤성 방광암(NMIBC) 환자는 BUB1의 중간 컷오프 값(BUB1 높음 및 BUB1 낮음)에 따라 두 그룹으로 분류하였다. 현재 실시간 PCR 코호트를 통한 단변량(Univariate) 및 다변량(multivariate) Cox 회귀 분석은 BUB1이 HR이 등급 시스템(HR, 4.629; 95% CI, 1.593-13.450; P=0.005)과 유사한 NMIBC 환자(HR, 4.642; 95 % CI, 1.021-21.097; P = 0.047)의 진행에 대한 독립적인 예측 변수가 될 수 있음을 보여주었다(표 3 참조).Survival analysis was performed to evaluate the prognostic power of BUB1 in non-muscle invasive bladder cancer (NMIBC). Patients with non-muscle invasive bladder cancer (NMIBC) were divided into two groups according to the median cutoff value of BUB1 (BUB1 high and BUB1 low). Univariate and multivariate Cox regression analyzes across the present real-time PCR cohort showed that BUB1 was associated with NMIBC patients (HR, 4.629; 95% CI, 1.593–13.450; P=0.005), with HR similar to the grading system (HR, 4.629; 95% CI, 1.593–13.450; P=0.005). 4.642; 95% CI, 1.021–21.097; P = 0.047) to be an independent predictor of progression (see Table 3).
Kaplan-Meier 분석에 따르면 낮은 50 번째 백분위 수에서 BUB1 발현 수준을 가진 NMIBC 환자는 높은 50 번째 백분위 수에서 발현 수준을 가진 환자보다 진행률이 더 낮은 것으로 나타났다(log-rank test, P = 0.007; 도 6a 참조). 더욱이 2004년 WHO 등급 시스템과 BUB1 발현을 결합했을 때 Kaplan-Meier 분석은 낮은 BUB1 발현을 가진 저등급(low grade) NMIBC 환자가 가장 낮은 진행을 경험한 반면, 높은 BUB1 발현을 가진 고등급(High grade) NMIBC 환자가 가장 많은 진행을 경험한 것으로 나타났다(log-rank test, P <0.0001; 도 6b 참조).According to Kaplan-Meier analysis, NMIBC patients with BUB1 expression levels in the lower 50th percentile had a lower progression rate than those with BUB1 expression levels in the upper 50th percentile (log-rank test, P = 0.007; Fig. 6a Reference). Moreover, when combining BUB1 expression with the 2004 WHO grading system, Kaplan-Meier analysis showed that patients with low-grade NMIBC with low BUB1 expression experienced the lowest progression, whereas high-grade NMIBC patients with high BUB1 expression experienced the lowest progression. ) NMIBC patients experienced the most progression (log-rank test, P <0.0001; see Fig. 6b).
<65 (Ref.) vs. >65Age
<65 (Ref.) vs. >65
Male (Ref.) vs. FemaleGender
Male (Ref.) vs. Male (Ref.) Female
≤1cm (Ref.) vs. 2-3cmTumor size
≤1cm (Ref.) vs. 2-3cm
Low (Ref.) vs. High2004 WHO Grade
Low (Ref.) vs. High
Ta (Ref.) vs. T1Stage
Ta (Ref.) vs. T1
No (Ref.) vs. YesBCG
No (Ref.) vs. Yes
High expression (Ref.) vs. Low expressionBUB1 expression
High expression (Ref.) vs. Low expression
BCG, Bacillus Calmette-Guerin; CI, confidence interval; HR, hazard ratio; Ref., reference.BCG, Bacillus Calmette-Guerin; CI, confidence interval; HR, hazard ratio; Ref., reference.
*P<0.05.*P<0.05.
검증 테스트verification test
비근침윤성 방광암(NMIBC)에서 BUB1의 발현 수준을 검증하기 위하여 공개된 방광암 환자 코호트 데이터(GSE13507 및 E-MTAT-4321)를 사용하였다.Published bladder cancer patient cohort data (GSE13507 and E-MTAT-4321) were used to verify the expression level of BUB1 in non-muscle invasive bladder cancer (NMIBC).
[GSE13507 데이터 세트의 BUB1 mRNA 발현: 내부 검증][BUB1 mRNA expression in the GSE13507 data set: internal validation]
BUB1 mRNA 발현은 GSE13507 코호트에서 정상 대조군과 비교하여 비근침윤성 방광암(NMIBC)에서 유의하게 향상되었으며(도 7a 참조), 이는 BUB1의 상향 조절이 비근침윤성 방광암(NMIBC) 발병에 중요한 사건이 될 수 있음을 나타낸다. BUB1 mRNA expression was significantly enhanced in non-muscle-invasive bladder cancer (NMIBC) compared to normal controls in the GSE13507 cohort (see Fig. 7a), suggesting that upregulation of BUB1 may be an important event in the development of non-muscle-invasive bladder cancer (NMIBC). indicate
한편, BUB1은 T1 단계의 비근침윤성 방광암(NMIBC) 환자에서 Ta 단계의 환자보다 상당히 과발현되어 있는 것으로 나타났으며, 저등급(low grade)의 비근침윤성 방광암(NMIBC)에 비해 고등급(High grade)의 비근침윤성 방광암(NMIBC)에서 더 높은 발현을 나타냈다(P <0.05) (도 7b 및 7c 참조). 질병의 생물학적 공격성을 나타내는 종양 단계 및 등급은 비근침윤성 방광암(NMIBC)의 재발 및 진행에 대한 중요한 예후 인자이기 때문에 이러한 결과는 BUB1의 상향 조절이 NMIBC 예후와 관련이 있을 수 있음을 시사한다.On the other hand, BUB1 was found to be significantly overexpressed in T1-stage non-muscle-invasive bladder cancer (NMIBC) patients than in Ta-stage patients, and high-grade compared to low-grade non-muscle-invasive bladder cancer (NMIBC). showed higher expression in non-muscle invasive bladder cancer (NMIBC) (P < 0.05) (see Figs. 7b and 7c). Since tumor stage and grade representing the biological aggressiveness of the disease are important prognostic factors for recurrence and progression of non-muscularly invasive bladder cancer (NMIBC), these results suggest that upregulation of BUB1 may be related to NMIBC prognosis.
단변량(Univariate) Cox 회귀 분석은 비근침윤성 방광암(NMIBC) 환자에서 BUB1의 발현이 무진행 생존(PFS)의 독립적인 예측인자임을 밝혀냈다. 단변량 분석에 따르면 BUB1 과발현 환자는 진행 위험이 15배 더 높은 것으로 나타났다(HR, 15; 95 % CI, 1.9-115; P = 0.011)(도 7d 참조).Univariate Cox regression analysis revealed that BUB1 expression was an independent predictor of progression-free survival (PFS) in patients with non-muscularly invasive bladder cancer (NMIBC). Univariate analysis showed that patients with BUB1 overexpression had a 15-fold higher risk of progression (HR, 15; 95% CI, 1.9-115; P = 0.011) (see Fig. 7d).
Kaplan Meier 생존 분석에 따르면 BUB1 발현이 더 높은 비근침윤성 방광암(NMIBC) 환자의 경우 무진행 생존율이 더 낮은 것으로 나타났다(log-rank test, P = 0.0009)(도 7e 참조).According to Kaplan Meier survival analysis, patients with non-muscularly invasive bladder cancer (NMIBC) with higher BUB1 expression showed a lower progression-free survival rate (log-rank test, P = 0.0009) (see FIG. 7e ).
[E-MTAT-4321 데이터 세트의 BUB1 mRNA 발현: 외부 검증][BUB1 mRNA expression in the E-MTAT-4321 data set: external validation]
유사한 결과로서, E-MTAT-4321 코호트에서 고등급(High grade) 또는 T1 단계의 비근침윤성 방광암(NMIBC) 환자가 저등급(low grade) 또는 Ta 단계의 환자보다 더 높은 BUB1 발현을 보인 것으로 확인되었다(모두 P <0.0001)(도 8a 및 8b 참조). 그러나 이 코호트에는 대조군이 없기 때문에 비근침윤성 방광암(NMIBC)과 대조군 간의 비교를 분석할 수 없었다.As a similar result, it was confirmed that in the E-MTAT-4321 cohort, patients with high grade or T1 non-muscle invasive bladder cancer (NMIBC) showed higher BUB1 expression than patients with low grade or Ta stage. (all P<0.0001) (see Figures 8A and 8B). However, comparisons between non-muscularly invasive bladder cancer (NMIBC) and controls could not be analyzed because there was no control group in this cohort.
단변량(Univariate) cox 회귀 분석에 따르면 BUB1 과발현 환자는 진행 위험이 5.7배 더 높은 것으로 나타나, 비근침윤성 방광암(NMIBC)에서 BUB1의 예후적 유의성을 나타냈다(HR, 5.7; 95% CI, 2.2-15; P <0.001)(도 8c 참조). Univariate cox regression analysis showed that patients with BUB1 overexpression had a 5.7-fold higher risk of progression, indicating prognostic significance of BUB1 in non-muscle-invasive bladder cancer (NMIBC) (HR, 5.7; 95% CI, 2.2-15 ; P<0.001) (see Figure 8c).
그리고 하위 50번째 백분위 수에 있는 BUB1 발현 수준을 가진 비근침윤성 방광암(NMIBC) 환자의 무진행 생존(PFS)은 상위 50번째 백분위 수에 있는 발현 수준을 가진 환자보다 진행을 덜 경험했다(log-rank test, P <0.00001) (도 8d 참조).And progression-free survival (PFS) of non-muscle invasive bladder cancer (NMIBC) patients with BUB1 expression levels in the lower 50th percentile experienced less progression than patients with expression levels in the upper 50th percentile (log-rank test, P < 0.00001) (see Fig. 8d).
종양 단계 및 등급을 E-MTAT-4321 코호트에서 BUB1 발현과 통합 할 때, BUB1의 발현이 높은 T1 단계 그룹의 비근침윤성 방광암(NMIBC) 환자는 최악의 무진행 생존(PFS)을 보였고, BUB1의 발현이 낮은 Ta 단계 그룹의 비근침윤성 방광암(NMIBC) 환자에서 가장 만족스러운 무진행 생존(PFS)을 보여주었다(log-rank test, P<0.0001)(도 8e 참조).When tumor stage and grade were integrated with BUB1 expression in the E-MTAT-4321 cohort, patients with non-muscle-invasive bladder cancer (NMIBC) in the T1 stage group with high BUB1 expression had the worst progression-free survival (PFS), and BUB1 expression Patients with non-muscular invasive bladder cancer (NMIBC) in this low Ta stage group showed the most satisfactory progression-free survival (PFS) (log-rank test, P<0.0001) (see Fig. 8e).
그리고 BUB1 발현이 높은 고등급(High grade) 그룹의 비근침윤성 방광암(NMIBC) 환자는 가장 힘든 무진행 생존(PFS)을 겪는 반면, BUB1 발현이 낮은 저등급(low grade) 그룹의 비근침윤성 방광암(NMIBC) 환자는 가장 낙관적인 무진행 생존(PFS)을 보여주었다(log-rank test, P <0.0001)(도 8f 참조).In addition, non-muscle invasive bladder cancer (NMIBC) patients in the high grade group with high BUB1 expression suffered the most difficult progression-free survival (PFS), whereas non-muscle invasive bladder cancer (NMIBC) in the low grade group with low BUB1 expression. ) patients showed the most optimistic progression-free survival (PFS) (log-rank test, P <0.0001) (see Fig. 8F).
BUB1 유전자를 넉다운시킨 방광암 세포주에서 암세포의 G2/M 세포주기 정지G2/M cell cycle arrest of cancer cells in bladder cancer cell lines in which the BUB1 gene was knocked down
BUB1이 방광암 세포에 어떠한 영향을 미치는지 확인하기 위해, 작은 간섭 RNA(siRNA)을 이용하여 BUB1 유전자를 넉다운(knockdown, KD)시킴으로써 방광암 세포주(5637 및 T24)에서 BUB1 발현을 조사하였다. 5637 및 T24 세포에서 BUB1의 발현량은 RT-qPCR를 통해 확인하였다. To confirm the effect of BUB1 on bladder cancer cells, BUB1 expression was examined in bladder cancer cell lines (5637 and T24) by knocking down (KD) the BUB1 gene using small interfering RNA (siRNA). The expression level of BUB1 in 5637 and T24 cells was confirmed by RT-qPCR.
BUB1의 발현 수준은 두 방광암 세포에서 비-표적 대조군(siNC) 대비 siBUB1을 형질주입한 세포에서 유의하게 감소하는 것으로 나타났으며(P <0.05), 넉다운 효율은 90-95%이었다(도 9a 및 9b 참조). 한편, 2개의 방광암 세포주의 세포주기 분석은 유세포 분석을 통해 수행되었으며, BUB1 siRNA로 형질주입된 세포의 경우 G2/M 단계에서 비율을 크게 증가한 것으로 나타났다(P <0.05)(도 9c 및 9d 참조). 따라서 BUB1 유전자 넉다운이 G2/M 단계의 전이를 억제할 수 있다. G2/M 단계에서 BUB1에 의해 유도된 세포주기의 비정상적인 조절은 세포의 과잉 증식과 비정상적인 암세포 수의 축적을 초래하여 결국 비근침윤성 방광암(NMIBC) 병인 및 진행으로 이어질수 있다고 사료된다.The expression level of BUB1 was found to be significantly decreased in cells transfected with siBUB1 compared to the non-target control (siNC) in both bladder cancer cells (P <0.05), and the knockdown efficiency was 90-95% (Fig. 9a and see 9b). On the other hand, cell cycle analysis of the two bladder cancer cell lines was performed through flow cytometry, and in the case of cells transfected with BUB1 siRNA, the ratio was significantly increased in the G2/M phase (P < 0.05) (see Figs. 9c and 9d). . Therefore, BUB1 gene knockdown can inhibit G2/M phase transition. It is thought that the abnormal regulation of the cell cycle induced by BUB1 in the G2/M phase can lead to cell overproliferation and accumulation of abnormal cancer cell numbers, eventually leading to the pathogenesis and progression of non-muscular invasive bladder cancer (NMIBC).
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 looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can 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 limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.
NMIBC: Non-Muscle Invasive Bladder Cancer
MIBC: Muscle invasive bladder cancer
NATs: Normal adjacent tissues
TURBT: Trans Urethral Resection of Bladder Tumor
BCG: Bacillus Calmette-Guerin
PFS: Progression-free survival
KD: knockdown
GO: gene ontology
DEGs: differential expressed genes
GSEA: gene set enrichment analysis
RT-qPCR: real-time quantitative reverse transcription polymerase chain reactionNMIBC: Non-Muscle Invasive Bladder Cancer
MIBC: Muscle invasive bladder cancer
NATs: Normal adjacent tissues
TURBT: Trans Urethral Resection of Bladder Tumor
BCG: Bacillus Calmette-Guerin
PFS: Progression-free survival
KD: knockdown
GO: gene ontology
DEGs: differentially expressed genes
GSEA: gene set enrichment analysis
RT-qPCR: real-time quantitative reverse transcription polymerase chain reaction
<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Use of BUB1 as a biomarker for predicting the prognosis of Non-muscle invasive bladder cancer <130> NPDC-96617 <160> 19 <170> KoPatentIn 3.0 <210> 1 <211> 3198 <212> DNA <213> Artificial Sequence <220> <223> BUB1 cDNA polynucleotide sequence <400> 1 atggacaccc cggaaaatgt ccttcaatac atacagtggg tagaagagaa ttttcctgag 60 aataaagaat acttgataac tttactagaa catttaatga aggaattttt agataagaag 120 aaataccaca atgacccaag attcatcagt tattgtttaa aatttgctga gtacaacagt 180 gacctccatc aattttttga gtttctgtac aaccatggga ttggaaccct gtcatcccct 240 ctgtacattg cctgggcggg gcatctggaa gcccaaggag agctgcagca tgccagtgct 300 gtccttcaga gaggaattca aaaccaggct gaacccagag agttcctgca acaacaatac 360 aggttatttc agacacgcct cactgaaacc catttgccag ctcaagctag aacctcagaa 420 cctctgcata atgttcaggt tttaaatcaa atgataacat caaaatcaaa tccaggaaat 480 aacatggcct gcatttctaa gaatcagggt tcagagcttt ctggagtgat atcttcagct 540 tgtgataaag agtcaaatat ggaacgaaga gtgatcacga tttctaaatc agaatattct 600 gtgcactcat ctttggcatc caaagttgat gttgagcagg ttgttatgta ttgcaaggag 660 aagcttattc gtggggaatc agaattttcc tttgaagaat tgagagccca gaaatacaat 720 caacggagaa agcatgagca atgggtaaat gaagacagac attatatgaa aaggaaagaa 780 gcaaatgctt ttgaagaaca gctattaaaa cagaaaatgg atgaacttca taagaagttg 840 catcaggtgg tggagacatc ccatgaggat ctgcccgctt cccaggaaag gtccgaggtt 900 aatccagcac gtatggggcc aagtgtaggc tcccagcagg aactgagagc gccatgtctt 960 ccagtaacct atcagcagac accagtgaac atggaaaaga acccaagaga ggcacctcct 1020 gttgttcctc ctttggcaaa tgctatttct gcagctttgg tgtccccagc caccagccag 1080 agcattgctc ctcctgttcc tttgaaagcc cagacagtaa cagactccat gtttgcagtg 1140 gccagcaaag atgctggatg tgtgaataag agtactcatg aattcaagcc acagagtgga 1200 gcagagatca aagaagggtg tgaaacacat aaggttgcca acacaagttc ttttcacaca 1260 actccaaaca catcactggg aatggttcag gcaacgccat ccaaagtgca gccatcaccc 1320 accgtgcaca caaaagaagc attaggtttc atcatgaata tgtttcaggc tcctacactt 1380 cctgatattt ctgatgacaa agatgaatgg caatctctag atcaaaatga agatgcattt 1440 gaagcccagt ttcaaaaaaa tgtaaggtca tctggggctt ggggagtcaa taagatcatc 1500 tcttctttgt catctgcttt tcatgtgttt gaagatggaa acaaagaaaa ttatggatta 1560 ccacagccta aaaataaacc cacaggagcc aggacctttg gagaacgctc tgtcagcaga 1620 cttccttcaa aaccaaagga ggaagtgcct catgctgaag agtttttgga tgactcaact 1680 gtatggggta ttcgctgcaa caaaaccctg gcacccagtc ctaagagccc aggagacttc 1740 acatctgctg cacaacttgc gtctacacca ttccacaagc ttccagtgga gtcagtgcac 1800 attttagaag ataaagaaaa tgtggtagca aaacagtgta cccaggcgac tttggattct 1860 tgtgaggaaa acatggtggt gccttcaagg gatggaaaat tcagtccaat tcaagagaaa 1920 agcccaaaac aggccttgtc gtctcacatg tattcagcat ccttacttcg tctgagccag 1980 cctgctgcag gtggggtact tacctgtgag gcagagttgg gcgttgaggc ttgcagactc 2040 acagacactg acgctgccat tgcagaagat ccaccagatg ctattgctgg gctccaagca 2100 gaatggatgc agatgagttc acttgggact gttgatgctc caaacttcat tgttgggaac 2160 ccatgggatg ataagctgat tttcaaactt ttatctgggc tttctaaacc agtgagttcc 2220 tatccaaata cttttgaatg gcaatgtaaa cttccagcca tcaagcccaa gactgaattt 2280 caattgggtt ctaagctggt ctatgtccat caccttcttg gagaaggagc ctttgcccag 2340 gtgtacgaag ctacccaggg agatctgaat gatgctaaaa ataaacagaa atttgtttta 2400 aaggtccaaa agcctgccaa cccctgggaa ttctacattg ggacccagtt gatggaaaga 2460 ctaaagccat ctatgcagca catgtttatg aagttctatt ctgcccactt attccagaat 2520 ggcagtgtat tagtaggaga gctctacagc tatggaacat tattaaatgc cattaacctc 2580 tataaaaata cccctgaaaa agtgatgcct caaggtcttg tcatctcttt tgctatgaga 2640 atgctttaca tgattgagca agtgcatgac tgtgaaatca ttcatggaga cattaaacca 2700 gacaatttca tacttggaaa cggatttttg gaacaggatg atgaagatga tttatctgct 2760 ggcttggcac tgattgacct gggtcagagt atagatatga aactttttcc aaaaggaact 2820 atattcacag caaagtgtga aacatctggt tttcagtgtg ttgagatgct cagcaacaaa 2880 ccatggaact accagatcga ttactttggg gttgctgcaa cagtatattg catgctcttt 2940 ggcacttaca tgaaagtgaa aaatgaagga ggagagtgta agcctgaagg tctttttaga 3000 aggcttcctc atttggatat gtggaatgaa ttttttcatg ttatgttgaa tattccagat 3060 tgtcatcatc ttccatcttt ggatttgtta aggcaaaagc tgaagaaagt atttcaacaa 3120 cactatacta acaagattag ggccctacgt aataggctaa ttgtactgct cttagaatgt 3180 aagcgttcac gaaaataa 3198 <210> 2 <211> 2382 <212> DNA <213> Artificial Sequence <220> <223> UHRF1 cDNA polynucleotide sequence <400> 2 atgtggatcc aggttcggac catggacggg aggcagaccc acacggtgga ctcgctgtcc 60 aggctgacca aggtggagga gctgaggcgg aagatccagg agctgttcca cgtggagcca 120 ggcctgcaga ggctgttcta caggggcaaa cagatggagg acggccatac cctcttcgac 180 tacgaggtcc gcctgaatga caccatccag ctcctggtcc gccagagcct cgtgctcccc 240 cacagcacca aggagcggga ctccgagctc tccgacaccg actccggctg ctgcctgggc 300 cagagtgagt cagacaagtc ctccacccac ggtgaggcgg ccgccgagac tgacagcagg 360 ccagccgatg aggacatgtg ggatgagacg gaattggggc tgtacaaggt caatgagtac 420 gtcgatgctc gggacacgaa catgggggcg tggtttgagg cgcaggtggt cagggtgacg 480 cggaaggccc cctcccggga cgagccctgc agctccacgt ccaggccggc gctggaggag 540 gacgtcattt accacgtgaa atacgacgac tacccggaga acggcgtggt ccagatgaac 600 tccagggacg tccgagcgcg cgcccgcacc atcatcaagt ggcaggacct ggaggtgggc 660 caggtggtca tgctcaacta caaccccgac aaccccaagg agcggggctt ctggtacgac 720 gcggagatct ccaggaagcg cgagaccagg acggcgcggg aactctacgc caacgtggtg 780 ctgggggatg attctctgaa cgactgtcgg atcatcttcg tggacgaagt cttcaagatt 840 gagcggccgg gtgaagggag ccccatggtt gacaacccca tgagacggaa gagcgggccg 900 tcctgcaagc actgcaagga cgacgtgaac agactctgcc gggtctgcgc ctgccacctg 960 tgcgggggcc ggcaggaccc cgacaagcag ctcatgtgcg atgagtgcga catggccttc 1020 cacatctact gcctggaccc gcccctcagc agtgttccca gcgaggacga gtggtactgc 1080 cctgagtgcc ggaatgatgc cagcgaggtg gtactggcgg gagagcggct gagagagagc 1140 aagaagaagg cgaagatggc ctcggccaca tcgtcctcac agcgggactg gggcaagggc 1200 atggcctgtg tgggccgcac caaggaatgt accatcgtcc cgtccaacca ctacggaccc 1260 atcccgggga tccccgtggg caccatgtgg cggttccgag tccaggtcag cgagtcgggt 1320 gtccatcggc cccacgtggc tggcatacac ggccggagca acgacggagc gtactcccta 1380 gtcctggcgg ggggctatga ggatgatgtg gaccatggga attttttcac atacacgggt 1440 agtggtggtc gagatctttc cggcaacaag aggaccgcgg aacagtcttg tgatcagaaa 1500 ctcaccaaca ccaacagggc gctggctctc aactgctttg ctcccatcaa tgaccaagaa 1560 ggggccgagg ccaaggactg gcggtcgggg aagccggtca gggtggtgcg caatgtcaag 1620 ggtggcaaga atagcaagta cgcccccgct gagggcaacc gctacgatgg catctacaag 1680 gttgtgaaat actggcccga gaaggggaag tccgggtttc tcgtgtggcg ctaccttctg 1740 cggagggacg atgatgagcc tggcccttgg acgaaggagg ggaaggaccg gatcaagaag 1800 ctggggctga ccatgcagta tccagaaggc tacctggaag ccctggccaa ccgagagcga 1860 gagaaggaga acagcaagag ggaggaggag gagcagcagg aggggggctt cgcgtccccc 1920 aggacgggca agggcaagtg gaagcggaag tcggcaggag gtggcccgag cagggccggg 1980 tccccgcgcc ggacatccaa gaaaaccaag gtggagccct acagtctcac ggcccagcag 2040 agcagcctca tcagagagga caagagcaac gccaagctgt ggaatgaggt cctggcgtca 2100 ctcaaggacc ggccggcgag cggcagcccg ttccagttgt tcctgagtaa agtggaggag 2160 acgttccagt gtatctgctg tcaggagctg gtgttccggc ccatcacgac cgtgtgccag 2220 cacaacgtgt gcaaggactg cctggacaga tcctttcggg cacaggtgtt cagctgccct 2280 gcctgccgct acgacctggg ccgcagctat gccatgcagg tgaaccagcc tctgcagacc 2340 gtcctcaacc agctcttccc cggctacggc aatggccggt ga 2382 <210> 3 <211> 912 <212> DNA <213> Artificial Sequence <220> <223> AURKB cDNA polynucleotide sequence <400> 3 atgagccgct ccaatgtcca gcccacagct gcccctggcc agaaggtgat ggagaatagc 60 agtgggacac ccgacatctt aacgcggcac ttcacaattg atgactttga gattgggcgt 120 cctctgggca aaggcaagtt tggaaacgtg tacttggctc gggagaagaa aagccatttc 180 atcgtggcgc tcaaggtcct cttcaagtcc cagatagaga aggagggcgt ggagcatcag 240 ctgcgcagag agatcgaaat ccaggcccac ctgcaccatc ccaacatcct gcgtctctac 300 aactattttt atgaccggag gaggatctac ttgattctag agtatgcccc ccgcggggag 360 ctctacaagg agctgcagaa gagctgcaca tttgacgagc agcgaacagc cacgatcatg 420 gaggagttgg cagatgctct aatgtactgc catgggaaga aggtgattca cagagacata 480 aagccagaaa atctgctctt agggctcaag ggagagctga agattgctga cttcggctgg 540 tctgtgcatg cgccctccct gaggaggaag acaatgtgtg gcaccctgga ctacctgccc 600 ccagagatga ttgaggggcg catgcacaat gagaaggtgg atctgtggtg cattggagtg 660 ctttgctatg agctgctggt ggggaaccca ccctttgaga gtgcatcaca caacgagacc 720 tatcgccgca tcgtcaaggt ggacctaaag ttccccgctt ccgtgcccat gggagcccag 780 gacctcatct ccaaactgct caggcataac ccctcggaac ggctgcccct ggcccaggtc 840 tcagcccacc cttgggtccg ggccaactct cggagggtgc tgcctccctc tgcccttcaa 900 tctgtcgcct ga 912 <210> 4 <211> 2242 <212> DNA <213> Artificial Sequence <220> <223> TPX2 cDNA polynucleotide sequence <400> 4 atgatgaagg agatactcaa aacatagatt catggtttga ggagaaggcc aatttggaga 60 ataagttact ggggaagaat ggaactggag ggctttttca gggcaaaact cctttgagaa 120 aggctaatct tcagcaagct attgtcacac ctttgaaacc agttgacaac acttactaca 180 aagaggcaga aaaagaaaat cttgtggaac aatccattcc gtcaaatgct tgttcttccc 240 tggaagttga ggcagccata tcaagaaaaa ctccagccca gcctcagaga agatctctta 300 ggctttctgc tcagaaggat ttggaacaga aagaaaagca tcatgtaaaa atgaaagcca 360 agagatgtgc cactcctgta atcatcgatg aaattctacc ctctaagaaa atgaaagttt 420 ctaacaacaa aaagaagcca gaggaagaag gcagtgctca tcaagatact gctgaaaaga 480 atgcatcttc cccagagaaa gccaagggta gacatactgt gccttgtatg ccacctgcaa 540 agcagaagtt tctaaaaagt actgaggagc aagagctgga gaagagtatg aaaatgcagc 600 aagaggtggt ggagatgcgg aaaaagaatg aagaattcaa gaaacttgct ctggctggaa 660 tagggcaacc tgtgaagaaa tcagtgagcc aggtcaccaa atcagttgac ttccacttcc 720 gcacagatga gcgaatcaaa caacatccta agaaccagga ggaatataag gaagtgaact 780 ttacatctga actacgaaag catccttcat ctcctgcccg agtgactaag ggatgtacca 840 ttgttaagcc tttcaacctg tcccaaggaa agaaaagaac atttgatgaa acagtttcta 900 catatgtgcc ccttgcacag caagttgaag acttccataa acgaacccct aacagatatc 960 atttgaggag caagaaggat gatattaacc tgttaccctc caaatcttct gtgaccaaga 1020 tttgcagaga cccacagact cctgtactgc aaaccaaaca ccgtgcacgg gctgtgacct 1080 gcaaaagtac agcagagctg gaggctgagg agctcgagaa attgcaacaa tacaaattca 1140 aagcacgtga acttgatccc agaatacttg aaggtgggcc catcttgccc aagaaaccac 1200 ctgtgaaacc acccaccgag cctattggct ttgatttgga aattgagaaa agaatccagg 1260 agcgagaatc aaagaagaaa acagaggatg aacactttga atttcattcc agaccttgcc 1320 ctactaagat tttggaagat gttgtgggtg ttcctgaaaa gaaggtactt ccaatcaccg 1380 tccccaagtc accagccttt gcattgaaga acagaattcg aatgcccacc aaagaagatg 1440 aggaagagga cgaaccggta gtgataaaag ctcaacctgt gccacattat ggggtgcctt 1500 ttaagcccca aatcccagag gcaagaactg tggaaatatg ccctttctcg tttgattctc 1560 gagacaaaga acgtcagtta cagaaggaga agaaaataaa agaactgcag aaaggggagg 1620 tgcccaagtt caaggcactt cccttgcctc attttgacac cattaacctg ccagagaaga 1680 aggtaaagaa tgtgacccag attgaacctt tctgcttgga gactgacaga agaggtgctc 1740 tgaaggcaca gacttggaag caccagctgg aagaagaact gagacagcag aaagaagcag 1800 cttgtttcaa ggctcgtcca aacaccgtca tctctcagga gccctttgtt cccaagaaag 1860 agaagaaatc agttgctgag ggcctttctg gttctctagt tcaggaacct tttcagctgg 1920 ctactgagaa gagagccaaa gagcggcagg agctggagaa gagaatggct gaggtagaag 1980 cccagaaagc ccagcagttg gaggaggcca gactacagga ggaagagcag aaaaaagagg 2040 agctggccag gctacggaga gaactggtgc ataaggcaaa tccaatacgc aagtaccagg 2100 gtctggagat aaagtcaagt gaccagcctc tgactgtgcc tgtatctccc aaattctcca 2160 ctcgattcca ctgctaaact cagctgtgag ctgcggatac cgcccggcaa tgggacctgc 2220 tcttaacctc aaacctagga cc 2242 <210> 5 <211> 3160 <212> DNA <213> Artificial Sequence <220> <223> BUB1B cDNA polynucleotide sequence <400> 5 atgcaggatg gcggcggtga agaaggaagg gggtgctctg agtgaagcca tgtccctgga 60 gggagatgaa tgggaactga gtaaagaaaa tgtacaacct ttaaggcaag ggcggatcat 120 gtccacgctt cagggagcac tggcacaaga atctgcctgt aacaatactc ttcagcagca 180 gaaacgggca tttgaatatg aaattcgatt ttacactgga aatgaccctc tggatgtttg 240 ggataggtat atcagctgga cagagcagaa ctatcctcaa ggtgggaagg agagtaatat 300 gtcaacgtta ttagaaagag ctgtagaagc actacaagga gaaaaacgat attatagtga 360 tcctcgattt ctcaatctct ggcttaaatt agggcgttta tgcaatgagc ctttggatat 420 gtacagttac ttgcacaacc aagggattgg tgtttcactt gctcagttct atatctcatg 480 ggcagaagaa tatgaagcta gagaaaactt taggaaagca gatgcgatat ttcaggaagg 540 gattcaacag aaggctgaac cactagaaag actacagtcc cagcaccgac aattccaagc 600 tcgagtgtct cggcaaactc tgttggcact tgagaaagaa gaagaggagg aagtttttga 660 gtcttctgta ccacaacgaa gcacactagc tgaactaaag agcaaaggga aaaagacagc 720 aagagctcca atcatccgtg taggaggtgc tctcaaggct ccaagccaga acagaggact 780 ccaaaatcca tttcctcaac agatgcaaaa taatagtaga attactgttt ttgatgaaaa 840 tgctgatgag gcttctacag cagagttgtc taagcctaca gtccagccat ggatagcacc 900 ccccatgccc agggccaaag agaatgagct gcaagcaggc ccttggaaca caggcaggtc 960 cttggaacac aggcctcgtg gcaatacagc ttcactgata gctgtacccg ctgtgcttcc 1020 cagtttcact ccatatgtgg aagagactgc acgacagcca gttatgacac catgtaaaat 1080 tgaacctagt ataaaccaca tcctaagcac cagaaagcct ggaaaggaag aaggagatcc 1140 tctacaaagg gttcagagcc atcagcaagc gtctgaggag aagaaagaga agatgatgta 1200 ttgtaaggag aagatttatg caggagtagg ggaattctcc tttgaagaaa ttcgggctga 1260 agttttccgg aagaaattaa aagagcaaag ggaagccgag ctattgacca gtgcagagaa 1320 gagagcagaa atgcagaaac agattgaaga gatggagaag aagctaaaag aaatccaaac 1380 tactcagcaa gaaagaacag gtgatcagca agaagagacg atgcctacaa aggagacaac 1440 taaactgcaa attgcttccg agtctcagaa aataccagga atgactctat ccagttctgt 1500 ttgtcaagta aactgttgtg ccagagaaac ttcacttgcg gagaacattt ggcaggaaca 1560 acctcattct aaaggtccca gtgtaccttt ctccattttt gatgagtttc ttctttcaga 1620 aaagaagaat aaaagtcctc ctgcagatcc cccacgagtt ttagctcaac gaagacccct 1680 tgcagttctc aaaacctcag aaagcatcac ctcaaatgaa gatgtgtctc cagatgtttg 1740 tgatgaattt acaggaattg aacccttgag cgaggatgcc attatcacag gcttcagaaa 1800 tgtaacaatt tgtcctaacc cagaagacac ttgtgacttt gccagagcag ctcgttttgt 1860 atccactcct tttcatgaga taatgtcctt gaaggatctc ccttctgatc ctgagagact 1920 gttaccggaa gaagatctag atgtaaagac ctctgaggac cagcagacag cttgtggcac 1980 tatctacagt cagactctca gcatcaagaa gctgagccca attattgaag acagtcgtga 2040 agccacacac tcctctggct tctctggttc ttctgcctcg gttgcaagca cctcctccat 2100 caaatgtctt caaattcctg agaaactaga acttactaat gagacttcag aaaaccctac 2160 tcagtcacca tggtgttcac agtatcgcag acagctactg aagtccctac cagagttaag 2220 tgcctctgca gagttgtgta tagaagacag accaatgcct aagttggaaa ttgagaagga 2280 aattgaatta ggtaatgagg attactgcat taaacgagaa tacctaatat gtgaagatta 2340 caagttattc tgggtggcgc caagaaactc tgcagaatta acagtaataa aggtatcttc 2400 tcaacctgtc ccatgggact tttatatcaa cctcaagtta aaggaacgtt taaatgaaga 2460 ttttgatcat ttttgcagct gttatcaata tcaagatggc tgtattgttt ggcaccaata 2520 tataaactgc ttcacccttc aggatcttct ccaacacagt gaatatatta cccatgaaat 2580 aacagtgttg attatttata accttttgac aatagtggag atgctacaca aagcagaaat 2640 agtccatggt gacttgagtc caaggtgtct gattctcaga aacagaatcc acgatcccta 2700 tgattgtaac aagaacaatc aagctttgaa gatagtggac ttttcctaca gtgttgacct 2760 tagggtgcag ctggatgttt ttaccctcag cggctttcgg actgtacaga tcctggaagg 2820 acaaaagatc ctggctaact gttcttctcc ctaccaggta gacctgtttg gtatagcaga 2880 tttagcacat ttactattgt tcaaggaaca cctacaggtc ttctgggatg ggtccttctg 2940 gaaacttagc caaaatattt ctgagctaaa agatggtgaa ttgtggaata aattctttgt 3000 gcggattctg aatgccaatg atgaggccac agtgtctgtt cttggggagc ttgcagcaga 3060 aatgaatggg gtttttgaca ctacattcca aagtcacctg aacaaagcct tatggaaggt 3120 agggaagtta actagtcctg gggctttgct ctttcagtga 3160 <210> 6 <211> 2673 <212> DNA <213> Artificial Sequence <220> <223> KIF20A cDNA polynucleotide sequence <400> 6 atgtcgcaag ggatcctttc tccgccagcg ggcttgctgt ccgatgacga tgtcgtagtt 60 tctcccatgt ttgagtccac agctgcagat ttggggtctg tggtacgcaa gaacctgcta 120 tcagactgct ctgtcgtctc tacctcccta gaggacaagc agcaggttcc atctgaggac 180 agtatggaga aggtgaaagt atacttgagg gttaggccct tgttaccttc agagttggaa 240 cgacaggaag atcagggttg tgtccgtatt gagaatgtgg agacccttgt tctacaagca 300 cccaaggact cttttgccct gaagagcaat gaacggggaa ttggccaagc cacacacagg 360 ttcacctttt cccagatctt tgggccagaa gtgggacagg catccttctt caacctaact 420 gtgaaggaga tggtaaagga tgtactcaaa gggcagaact ggctcatcta tacatatgga 480 gtcactaact cagggaaaac ccacacgatt caaggtacca tcaaggatgg agggattctc 540 ccccggtccc tggcgctgat cttcaatagc ctccaaggcc aacttcatcc aacacctgat 600 ctgaagccct tgctctccaa tgaggtaatc tggctagaca gcaagcagat ccgacaggag 660 gaaatgaaga agctgtccct gctaaatgga ggcctccaag aggaggagct gtccacttcc 720 ttgaagagga gtgtctacat cgaaagtcgg ataggtacca gcaccagctt cgacagtggc 780 attgctgggc tctcttctat cagtcagtgt accagcagta gccagctgga tgaaacaagt 840 catcgatggg cacagccaga cactgcccca ctacctgtcc cggcaaacat tcgcttctcc 900 atctggatct cattctttga gatctacaac gaactgcttt atgacctatt agaaccgcct 960 agccaacagc gcaagaggca gactttgcgg ctatgcgagg atcaaaatgg caatccctat 1020 gtgaaagatc tcaactggat tcatgtgcaa gatgctgagg aggcctggaa gctcctaaaa 1080 gtgggtcgta agaaccagag ctttgccagc acccacctca accagaactc cagccgcagt 1140 cacagcatct tctcaatcag gatcctacac cttcaggggg aaggagatat agtccccaag 1200 atcagcgagc tgtcactctg tgatctggct ggctcagagc gctgcaaaga tcagaagagt 1260 ggtgaacggt tgaaggaagc aggaaacatt aacacctctc tacacaccct gggccgctgt 1320 attgctgccc ttcgtcaaaa ccagcagaac cggtcaaagc agaacctggt tcccttccgt 1380 gacagcaagt tgactcgagt gttccaaggt ttcttcacag gccgaggccg ttcctgcatg 1440 attgtcaatg tgaatccctg tgcatctacc tatgatgaaa ctcttcatgt ggccaagttc 1500 tcagccattg ctagccagct tgtgcatgcc ccacctatgc aactgggatt cccatccctg 1560 cactcgttca tcaaggaaca tagtcttcag gtatccccca gcttagagaa aggggctaag 1620 gcagacacag gccttgatga tgatattgaa aatgaagctg acatctccat gtatggcaaa 1680 gaggagctcc tacaagttgt ggaagccatg aagacactgc ttttgaagga acgacaggaa 1740 aagctacagc tggagatgca tctccgagat gaaatttgca atgagatggt agaacagatg 1800 caacagcggg aacagtggtg cagtgaacat ttggacaccc aaaaggaact attggaggaa 1860 atgtatgaag aaaaactaaa tatcctcaag gagtcactga caagttttta ccaagaagag 1920 attcaggagc gggatgaaaa gattgaagag ctagaagctc tcttgcagga agccagacaa 1980 cagtcagtgg cccatcagca atcagggtct gaattggccc tacggcggtc acaaaggttg 2040 gcagcttctg cctccaccca gcagcttcag gaggttaaag ctaaattaca gcagtgcaaa 2100 gcagagctaa actctaccac tgaagagttg cataagtatc agaaaatgtt agaaccacca 2160 ccctcagcca agcccttcac cattgatgtg gacaagaagt tagaagaggg ccagaagaat 2220 ataaggctgt tgcggacaga gcttcagaaa cttggtgagt ctctccaatc agcagagaga 2280 gcttgttgcc acagcactgg ggcaggaaaa cttcgtcaag ccttgaccac ttgtgatgac 2340 atcttaatca aacaggacca gactctggct gaactgcaga acaacatggt gctagtgaaa 2400 ctggaccttc ggaagaaggc agcatgtatt gctgagcagt atcatactgt gttgaaactc 2460 caaggccagg tttctgccaa aaagcgcctt ggtaccaacc aggaaaatca gcaaccaaac 2520 caacaaccac cagggaagaa accattcctt cgaaatttac ttccccgaac accaacctgc 2580 caaagctcaa cagactgcag cccttatgcc cggatcctac gctcacggcg ttccccttta 2640 ctcaaatctg ggccttttgg caaaaagtac taa 2673 <210> 7 <211> 1500 <212> DNA <213> Artificial Sequence <220> <223> CDC20 cDNA polynucleotide sequence <400> 7 atggcacagt tcgcgttcga gagtgacctg cactcgctgc ttcagctgga tgcacccatc 60 cccaatgcac cccctgcgcg ctggcagcgc aaagccaagg aagccgcagg cccggccccc 120 tcacccatgc gggccgccaa ccgatcccac agcgccggca ggactccggg ccgaactcct 180 ggcaaatcca gttccaaggt tcagaccact cctagcaaac ctggcggtga ccgctatatc 240 ccccatcgca gtgctgccca gatggaggtg gccagcttcc tcctgagcaa ggagaaccag 300 cctgaaaaca gccagacgcc caccaagaag gaacatcaga aagcctgggc tttgaacctg 360 aacggttttg atgtagagga agccaagatc cttcggctca gtggaaaacc acaaaatgcg 420 ccagagggtt atcagaacag actgaaagta ctctacagcc aaaaggccac tcctggctcc 480 agccggaaga cctgccgtta cattccttcc ctgccagacc gtatcctgga tgcgcctgaa 540 atccgaaatg actattacct gaaccttgtg gattggagtt ctgggaatgt actggccgtg 600 gcactggaca acagtgtgta cctgtggagt gcaagctctg gtgacatcct gcagcttttg 660 caaatggagc agcctgggga atatatatcc tctgtggcct ggatcaaaga gggcaactac 720 ttggctgtgg gcaccagcag tgctgaggtg cagctatggg atgtgcagca gcagaaacgg 780 cttcgaaata tgaccagtca ctctgcccga gtgggctccc taagctggaa cagctatatc 840 ctgtccagtg gttcacgttc tggccacatc caccaccatg atgttcgggt agcagaacac 900 catgtggcca cactgagtgg ccacagccag gaagtgtgtg ggctgcgctg ggccccagat 960 ggacgacatt tggccagtgg tggtaatgat aacttggtca atgtgtggcc tagtgctcct 1020 ggagagggtg gctgggttcc tctgcagaca ttcacccagc atcaaggggc tgtcaaggcc 1080 gtagcatggt gtccctggca gtccaatgtc ctggcaacag gagggggcac cagtgatcga 1140 cacattcgca tctggaatgt gtgctctggg gcctgtctga gtgccgtgga tgcccattcc 1200 caggtgtgct ccatcctctg gtctccccat tacaaggagc tcatctcagg ccatggcttt 1260 gcacagaacc agctagttat ttggaagtac ccaaccatgg ccaaggtggc tgaactcaaa 1320 ggtcacacat cccgggtcct gagtctgacc atgagcccag atggggccac agtggcatcc 1380 gcagcagcag atgagaccct gaggctatgg cgctgttttg agttggaccc tgcgcggcgg 1440 cgggagcggg agaaggccag tgcagccaaa agcagcctca tccaccaagg catccgctga 1500 1500 <210> 8 <211> 543 <212> DNA <213> Artificial Sequence <220> <223> TK1 cDNA polynucleotide sequence <400> 8 atgagctgca ttaacctgcc cactgtgctg cctggctccc ccagcaagac ccgggggcag 60 atccaggtga ttctcgggcc gatgttctca ggaaaaagca cagagttgat gagacgcgtc 120 cgtcgcttcc agattgctca gtacaagtgc ctggtgatca agtatgccaa agacactcgc 180 tacagcagca gcttctgcac acatgaccgg aacaccatgg aggcactgcc cgcctgcctg 240 ctccgagacg tggcccagga ggccctgggc gtggctgtca taggcatcga cgaggggcag 300 tttttccctg acatcgtgga gttctgcgag gccatggcca acgccgggaa gaccgtaatt 360 gtggctgcac tggatgggac cttccagagg aagccatttg gggccatcct gaacctggtg 420 ccgctggccg agagcgtggt gaagctgacg gcggtgtgca tggagtgctt ccgggaagcc 480 gcctatacca agaggctcgg cacagagaag gagcagattc tgcaatgcag ccctgccaac 540 tga 543 <210> 9 <211> 1395 <212> DNA <213> Artificial Sequence <220> <223> CEP55 cDNA polynucleotide sequence <400> 9 atgtcttcca gaagtaccaa agatttaatt aaaagtaagt ggggatcgaa gcctagtaac 60 tccaaatccg aaactacatt agaaaaatta aagggagaaa ttgcacactt aaagacatca 120 gtggatgaaa tcacaagtgg gaaaggaaag ctgactgata aagagagaca cagacttttg 180 gagaaaattc gagtccttga ggctgagaag gagaagaatg cttatcaact cacagagaag 240 gacaaagaaa tacagcgact gagagaccaa ctgaaggcca gatatagtac taccacattg 300 cttgaacagc tggaagagac aacgagagaa ggagaaagga gggagcaggt gttgaaagcc 360 ttatctgaag agaaagacgt attgaaacaa cagttgtctg ctgcaacctc acgaattgct 420 gaacttgaaa gcaaaaccaa tacactccgt ttatcacaga ctgtggctcc aaactgcttc 480 aactcatcaa taaataatat tcatgaaatg gaaatacagc tgaaagatgc tctggagaaa 540 aatcagcagt ggctcgtgta tgatcagcag cgggaagtct atgtaaaagg acttttagca 600 aagatctttg agttggaaaa gaaaacggaa acagctgctc attcactccc acagcagaca 660 aaaaagcctg aatcagaagg ttatcttcaa gaagagaagc agaaatgtta caacgatctc 720 ttggcaagtg caaaaaaaga tcttgaggtt gaacgacaaa ccataactca gctgagtttt 780 gaactgagtg aatttcgaag aaaatatgaa gaaacccaaa aagaagttca caatttaaat 840 cagctgttgt attcacaaag aagggcagat gtgcaacatc tggaagatga taggcataaa 900 acagagaaga tacaaaaact cagggaagag aatgatattg ctaggggaaa acttgaagaa 960 gagaagaaga gatccgaaga gctcttatct caggtccagt ttctttacac atctctgcta 1020 aagcagcaag aagaacaaac aagggtagct ctgttggaac aacagatgca ggcatgtact 1080 ttagactttg aaaatgaaaa actcgaccgt caacatgtgc agcatcaatt gcatgtaatt 1140 cttaaggagc tccgaaaagc aagaaatcaa ataacacagt tggaatcctt gaaacagctt 1200 catgagtttg ccatcacaga gccattagtc actttccaag gagagactga aaacagagaa 1260 aaagttgccg cctcaccaaa aagtcccact gctgcactca atgaaagcct ggtggaatgt 1320 cccaagtgca atatacagta tccagccact gagcatcgcg atctgcttgt ccatgtggaa 1380 tactgttcaa agtag 1395 <210> 10 <211> 2571 <212> DNA <213> Artificial Sequence <220> <223> TTK cDNA polynucleotide sequence <400> 10 atggaatccg aggatttaag tggcagagaa ttgacaattg attccataat gaacaaagtg 60 agagacatta aaaataagtt taaaaatgaa gaccttactg atgaactaag cttgaataaa 120 atttctgctg atactacaga taactcggga actgttaacc aaattatgat gatggcaaac 180 aacccagagg actggttgag tttgttgctc aaactagaga aaaacagtgt tccgctaagt 240 gatgctcttt taaataaatt gattggtcgt tacagtcaag caattgaagc gcttccccca 300 gataaatatg gccaaaatga gagttttgct agaattcaag tgagatttgc tgaattaaaa 360 gctattcaag agccagatga tgcacgtgac tactttcaaa tggccagagc aaactgcaag 420 aaatttgctt ttgttcatat atcttttgca caatttgaac tgtcacaagg taatgtcaaa 480 aaaagtaaac aacttcttca aaaagctgta gaacgtggag cagtaccact agaaatgctg 540 gaaattgccc tgcggaattt aaacctccaa aaaaagcagc tgctttcaga ggaggaaaag 600 aagaatttat cagcatctac ggtattaact gcccaagaat cattttccgg ttcacttggg 660 catttacaga ataggaacaa cagttgtgat tccagaggac agactactaa agccaggttt 720 ttatatggag agaacatgcc accacaagat gcagaaatag gttaccggaa ttcattgaga 780 caaactaaca aaactaaaca gtcatgccca tttggaagag tcccagttaa ccttctaaat 840 agcccagatt gtgatgtgaa gacagatgat tcagttgtac cttgttttat gaaaagacaa 900 acctctagat cagaatgccg agatttggtt gtgcctggat ctaaaccaag tggaaatgat 960 tcctgtgaat taagaaattt aaagtctgtt caaaatagtc atttcaagga acctctggtg 1020 tcagatgaaa agagttctga acttattatt actgattcaa taaccctgaa gaataaaacg 1080 gaatcaagtc ttctagctaa attagaagaa actaaagagt atcaagaacc agaggttcca 1140 gagagtaacc agaaacagtg gcaatctaag agaaagtcag agtgtattaa ccagaatcct 1200 gctgcatctt caaatcactg gcagattccg gagttagccc gaaaagttaa tacagagaaa 1260 cataccactt ttgagcaacc tgtcttttca gtttcaaaac agtcaccacc aatatcaaca 1320 tctaaatggt ttgacccaaa atctatttgt aagacaccaa gcagcaatac cttggatgat 1380 tacatgagct gttttagaac tccagttgta aagaatgact ttccacctgc ttgtcagttg 1440 tcaacacctt atggccaacc tgcctgtttc cagcagcaac agcatcaaat acttgccact 1500 ccacttcaaa atttacaggt tttagcatct tcttcagcaa atgaatgcat ttcggttaaa 1560 ggaagaattt attccatatt aaagcagata ggaagtggag gttcaagcaa ggtatttcag 1620 gtgttaaatg aaaagaaaca gatatatgct ataaaatatg tgaacttaga agaagcagat 1680 aaccaaactc ttgatagtta ccggaacgaa atagcttatt tgaataaact acaacaacac 1740 agtgataaga tcatccgact ttatgattat gaaatcacgg accagtacat ctacatggta 1800 atggagtgtg gaaatattga tcttaatagt tggcttaaaa agaaaaaatc cattgatcca 1860 tgggaacgca agagttactg gaaaaatatg ttagaggcag ttcacacaat ccatcaacat 1920 ggcattgttc acagtgatct taaaccagct aactttctga tagttgatgg aatgctaaag 1980 ctaattgatt ttgggattgc aaaccaaatg caaccagata caacaagtgt tgttaaagat 2040 tctcaggttg gcacagttaa ttatatgcca ccagaagcaa tcaaagatat gtcttcctcc 2100 agagagaatg ggaaatctaa gtcaaagata agccccaaaa gtgatgtttg gtccttagga 2160 tgtattttgt actatatgac ttacgggaaa acaccatttc agcagataat taatcagatt 2220 tctaaattac atgccataat tgatcctaat catgaaattg aatttcccga tattccagag 2280 aaagatcttc aagatgtgtt aaagtgttgt ttaaaaaggg acccaaaaca gaggatatcc 2340 attcctgagc tcctggctca tccatatgtt caaattcaaa ctcatccagt taaccaaatg 2400 gccaagggaa ccactgaaga aatgaaatat gttctgggcc aacttgttgg tctgaattct 2460 cctaactcca ttttgaaagc tgctaaaact ttatatgaac actatagtgg tggtgaaagt 2520 cataattctt catcctccaa gacttttgaa aaaaaaaggg gaaaaaaatg a 2571 <210> 11 <211> 1197 <212> DNA <213> Artificial Sequence <220> <223> CCNB2 cDNA polynucleotide sequence <400> 11 atggcgctgc tccgacgccc gacggtgtcc agtgatttgg agaatattga cacaggagtt 60 aattctaaag ttaagagtca tgtgactatt aggcgaactg ttttagaaga aattggaaat 120 agagttacaa ccagagcagc acaagtagct aagaaagctc agaacaccaa agttccagtt 180 caacccacca aaacaacaaa tgtcaacaaa caactgaaac ctactgcttc tgtcaaacca 240 gtacagatgg aaaagttggc tccaaagggt ccttctccca cacctgagga tgtctccatg 300 aaggaagaga atctctgcca agctttttct gatgccttgc tctgcaaaat cgaggacatt 360 gataacgaag attgggagaa ccctcagctc tgcagtgact acgttaagga tatctatcag 420 tatctcaggc agctggaggt tttgcagtcc ataaacccac atttcttaga tggaagagat 480 ataaatggac gcatgcgtgc catcctagtg gattggctgg tacaagtcca ctccaagttt 540 aggcttctgc aggagactct gtacatgtgc gttggcatta tggatcgatt tttacaggtt 600 cagccagttt cccggaagaa gcttcaatta gttgggatta ctgctctgct cttggcttcc 660 aagtatgagg agatgttttc tccaaatatt gaagactttg tttacatcac agacaatgct 720 tataccagtt cccaaatccg agaaatggaa actctaattt tgaaagaatt gaaatttgag 780 ttgggtcgac ccttgccact acacttctta aggcgagcat caaaagccgg ggaggttgat 840 gttgaacagc acactttagc caagtatttg atggagctga ctctcatcga ctatgatatg 900 gtgcattatc atccttctaa ggtagcagca gctgcttcct gcttgtctca gaaggttcta 960 ggacaaggaa aatggaactt aaagcagcag tattacacag gatacacaga gaatgaagta 1020 ttggaagtca tgcagcacat ggccaagaat gtggtgaaag taaatgaaaa cttaactaaa 1080 ttcatcgcca tcaagaataa gtatgcaagc agcaaactcc tgaagatcag catgatccct 1140 cagctgaact caaaagccgt caaagacctt gcctccccac tgataggaag gtcctag 1197 <210> 12 <211> 2085 <212> DNA <213> Artificial Sequence <220> <223> HJURP cDNA polynucleotide sequence <400> 12 atgctgggta cgctgcgcgc catggagggc gaggacgtgg aagacgacca gctgctgcag 60 aagctcaggg ccagtcgccg ccgcttccag aggcgcatgc agcggctgat agagaagtac 120 aaccagccct tcgaggacac cccggtggtg caaatggcca cgctgaccta cgagacgcca 180 cagggattga gaatttgggg tggaagacta ataaaggaaa gaaacgaagg agagatccag 240 cctgcagtgc ctcaaagccc tttgaaaaat gaattaagaa ggaaatactt gacccaagtg 300 gatatactgc tacaaggtgc agagtatttt gagtgtgcag gtaacagagc tggaagggat 360 gtacgtgtga ctccgctgcc ttcactggcc tcacctgccg tgcctgcccc cggatactgc 420 agtcgtatct ccagaaagag tcctggtgac ccagcgaaac cagcttcatc tcccagagaa 480 tgggatcctt tgcatccttc ctccacagac atggccttag tacctagaaa tgacagcctc 540 tccctacaag agaccagtag cagcagcttc ttaagcagcc agccctttga agatgatgac 600 atttgcaatg tgaccatcag tgacctgtac gcagggatgc tgcactccat gagccggctg 660 ttgagcacaa agccatcaag catcatctcc accaaaacgt tcatcatgca aaactggaac 720 tccaggagga ggcacagata taagagcagg atgaacaaaa catattgcaa aggagccaga 780 cgttctcaga ggagctccaa ggagaacttc ataccctgct ctgagcctgt gaaagggaca 840 ggggcattaa gagattgcaa gaacgtatta gatgtttctt gccgtaagac aggtttaaaa 900 ttggaaaaag cttttcttga agtcaacaga ccccaaatcc ataagttaga tccaagttgg 960 aaggagcgca aagtgacacc ctcgaagtat tcttccttga tttacttcga ctccagtgca 1020 acatataatc ttgatgagga aaatagattt aggacattaa aatggttaat ttctcctgta 1080 aaaatagttt ccagaccaac aatacgacag ggccatggag agaaccgtca gagggagatt 1140 gaaatccgat ttgatcagct tcatcgggaa tattgcctga gtcccaggaa ccagcctcgc 1200 cggatgtgcc tcccggactc ctgggccatg aacatgtaca gagggggtcc tgcgagtcct 1260 ggtggccttc agggcttaga aacccgcagg ctgagtttac cttccagcaa agcaaaagca 1320 aaaagtttaa gtgaggcttt tgaaaaccta ggcaaaagat ctctggaagc aggtaggtgc 1380 ctgcccaaga gcgattcatc ttcatcactt ccaaagacca accccacaca cagcgcaact 1440 cgcccgcagc agacatctga ccttcacgtt cagggaaata gttctggaat atttagaaag 1500 tcagtgtcac ccagcaaaac tctttcagtc ccagataaag aagtgccagg ccacggaagg 1560 aatcgttacg atgaaattaa agaagaattt gacaagcttc atcaaaagta ttgcctcaaa 1620 tctcctgggc agatgacagt gcctttatgt attggagtgt ctacagataa agcaagtatg 1680 gaagttcgat atcaaacaga aggcttctta ggaaaattaa atccagaccc tcacttccag 1740 ggtttccaga agttgccatc atcacccctg gggtgcagaa aaagtctact gggctcaact 1800 gcaattgagg ctccttcatc tacatgtgtt gctcgtgcca tcacgaggga tggcacgagg 1860 gaccatcagt tccctgcaaa aagacccagg ctatcagaac cccagggctc cggacgccag 1920 ggcaattccc tgggtgcctc agatggggtg gacaacaccg tcagaccggg agaccagggc 1980 agctcttcac agcccaactc agaagagaga ggagagaaca cgtcttacag gatggaagag 2040 aaaagtgatt tcatgctaga aaaattggaa actaaaagtg tgtag 2085 <210> 13 <211> 1320 <212> DNA <213> Artificial Sequence <220> <223> NUSAP1 cDNA polynucleotide sequence <400> 13 atgatcatcc cctctctaga ggagctggac tccctcaagt acagtgacct gcagaactta 60 gccaagagtc tgggtctccg ggccaacctg agggcaacca agttgttaaa agccttgaaa 120 ggctacatta aacatgaggc aagaaaagga aatgagaatc aggatgaaag tcaaacttct 180 gcatcctctt gtgatgagac tgagatacag atcagcaacc aggaagaagc tgagagacag 240 ccacttggcc atgtcaccaa aacaaggaga aggtgcaaga ctgtccgtgt ggaccctgac 300 tcacagaatc attcagagat aaaaataagt aatcccactg aattccagaa tcatgaaaag 360 caggaaagcc aggatctcag agctactgca aaagttcctt ctccaccaga cgagcaccaa 420 gaagctgaga atgctgtttc ctcaggtaac agagattcaa aggtaccttc agaaggaaag 480 aaatctctct acacagatga gtcatccaaa cctggaaaaa ataaaagaac tgcaatcact 540 actccaaact ttaagaagct tcatgaagct cattttaagg aaatggagtc cattgatcaa 600 tatattgaga gaaaaaagaa acattttgaa gaacacaatt ccatgaatga actgaagcag 660 cccatcaata agggaggggt caggactcca gtacctccaa gaggaagact ctctgtggct 720 tctactccca tcagccaacg acgctcgcaa ggccggtctt gtggccctgc aagtcagagt 780 accttgggtc tgaaggggtc actcaagcgc tctgctatct ctgcagctaa aacgggtgtc 840 aggttttcag ctgctactaa agataatgag cataagcgtt cactgaccaa gactccagcc 900 agaaagtctg cacatgtgac cgtgtctggg ggcaccccaa aaggcgaggc tgtgcttggg 960 acacacaaat taaagaccat cacggggaat tctgctgctg ttattacccc attcaagttg 1020 acaactgagg caacgcagac tccagtctcc aataagaaac cagtgtttga tcttaaagca 1080 agtttgtctc gtcccctcaa ctatgaacca cacaaaggaa agctaaaacc atgggggcaa 1140 tctaaagaaa ataattatct aaatcaacat gtcaacagaa ttaacttcta caagaaaact 1200 tacaaacaac cccatctcca gacaaaggaa gagcaacgga agaaacgcga gcaagaacga 1260 aaggagaaga aagcaaaggt tttgggaatg cgaaggggcc tcattttggc tgaagattaa 1320 1320 <210> 14 <211> 4596 <212> DNA <213> Artificial Sequence <220> <223> TOP2A cDNA polynucleotide sequence <400> 14 atggaagtgt caccattgca gcctgtaaat gaaaatatgc aagtcaacaa aataaagaaa 60 aatgaagatg ctaagaaaag actgtctgtt gaaagaatct atcaaaagaa aacacaattg 120 gaacatattt tgctccgccc agacacctac attggttctg tggaattagt gacccagcaa 180 atgtgggttt acgatgaaga tgttggcatt aactataggg aagtcacttt tgttcctggt 240 ttgtacaaaa tctttgatga gattctagtt aatgctgcgg acaacaaaca aagggaccca 300 aaaatgtctt gtattagagt cacaattgat ccggaaaaca atttaattag tatatggaat 360 aatggaaaag gtattcctgt tgttgaacac aaagttgaaa agatgtatgt cccagctctc 420 atatttggac agctcctaac ttctagtaac tatgatgatg atgaaaagaa agtgacaggt 480 ggtcgaaatg gctatggagc caaattgtgt aacatattca gtaccaaatt tactgtggaa 540 acagccagta gagaatacaa gaaaatgttc aaacagacat ggatggataa tatgggaaga 600 gctggtgaga tggaactcaa gcccttcaat ggagaagatt atacatgtat cacctttcag 660 cctgatttgt ctaagtttaa aatgcaaagc ctggacaaag atattgttgc actaatggtc 720 agaagagcat atgatattgc tggatccacc aaagatgtca aagtctttct taatggaaat 780 aaactgccag taaaaggatt tcgtagttat gtggacatgt atttgaagga caagttggat 840 gaaactggta actccttgaa agtaatacat gaacaagtaa accacaggtg ggaagtgtgt 900 ttaactatga gtgaaaaagg ctttcagcaa attagctttg tcaacagcat tgctacatcc 960 aagggtggca gacatgttga ttatgtagct gatcagattg tgactaaact tgttgatgtt 1020 gtgaagaaga agaacaaggg tggtgttgca gtaaaagcac atcaggtgaa aaatcacatg 1080 tggatttttg taaatgcctt aattgaaaac ccaacctttg actctcagac aaaagaaaac 1140 atgactttac aacccaagag ctttggatca acatgccaat tgagtgaaaa atttatcaaa 1200 gctgccattg gctgtggtat tgtagaaagc atactaaact gggtgaagtt taaggcccaa 1260 gtccagttaa acaagaagtg ttcagctgta aaacataata gaatcaaggg aattcccaaa 1320 ctcgatgatg ccaatgatgc agggggccga aactccactg agtgtacgct tatcctgact 1380 gagggagatt cagccaaaac tttggctgtt tcaggccttg gtgtggttgg gagagacaaa 1440 tatggggttt tccctcttag aggaaaaata ctcaatgttc gagaagcttc tcataagcag 1500 atcatggaaa atgctgagat taacaatatc atcaagattg tgggtcttca gtacaagaaa 1560 aactatgaag atgaagattc attgaagacg cttcgttatg ggaagataat gattatgaca 1620 gatcaggacc aagatggttc ccacatcaaa ggcttgctga ttaattttat ccatcacaac 1680 tggccctctc ttctgcgaca tcgttttctg gaggaattta tcactcccat tgtaaaggta 1740 tctaaaaaca agcaagaaat ggcattttac agccttcctg aatttgaaga gtggaagagt 1800 tctactccaa atcataaaaa atggaaagtc aaatattaca aaggtttggg caccagcaca 1860 tcaaaggaag ctaaagaata ctttgcagat atgaaaagac atcgtatcca gttcaaatat 1920 tctggtcctg aagatgatgc tgctatcagc ctggccttta gcaaaaaaca gatagatgat 1980 cgaaaggaat ggttaactaa tttcatggag gatagaagac aacgaaagtt acttgggctt 2040 cctgaggatt acttgtatgg acaaactacc acatatctga catataatga cttcatcaac 2100 aaggaactta tcttgttctc aaattctgat aacgagagat ctatcccttc tatggtggat 2160 ggtttgaaac caggtcagag aaaggttttg tttacttgct tcaaacggaa tgacaagcga 2220 gaagtaaagg ttgcccaatt agctggatca gtggctgaaa tgtcttctta tcatcatggt 2280 gagatgtcac taatgatgac cattatcaat ttggctcaga attttgtggg tagcaataat 2340 ctaaacctct tgcagcccat tggtcagttt ggtaccaggc tacatggtgg caaggattct 2400 gctagtccac gatacatctt tacaatgctc agctctttgg ctcgattgtt atttccacca 2460 aaagatgatc acacgttgaa gtttttatat gatgacaacc agcgtgttga gcctgaatgg 2520 tacattccta ttattcccat ggtgctgata aatggtgctg aaggaatcgg tactgggtgg 2580 tcctgcaaaa tccccaactt tgatgtgcgt gaaattgtaa ataacatcag gcgtttgatg 2640 gatggagaag aacctttgcc aatgcttcca agttacaaga acttcaaggg tactattgaa 2700 gaactggctc caaatcaata tgtgattagt ggtgaagtag ctattcttaa ttctacaacc 2760 attgaaatct cagagcttcc cgtcagaaca tggacccaga catacaaaga acaagttcta 2820 gaacccatgt tgaatggcac cgagaagaca cctcctctca taacagacta tagggaatac 2880 catacagata ccactgtgaa atttgttgtg aagatgactg aagaaaaact ggcagaggca 2940 gagagagttg gactacacaa agtcttcaaa ctccaaacta gtctcacatg caactctatg 3000 gtgctttttg accacgtagg ctgtttaaag aaatatgaca cggtgttgga tattctaaga 3060 gacttttttg aactcagact taaatattat ggattaagaa aagaatggct cctaggaatg 3120 cttggtgctg aatctgctaa actgaataat caggctcgct ttatcttaga gaaaatagat 3180 ggcaaaataa tcattgaaaa taagcctaag aaagaattaa ttaaagttct gattcagagg 3240 ggatatgatt cggatcctgt gaaggcctgg aaagaagccc agcaaaaggt tccagatgaa 3300 gaagaaaatg aagagagtga caacgaaaag gaaactgaaa agagtgactc cgtaacagat 3360 tctggaccaa ccttcaacta tcttcttgat atgccccttt ggtatttaac caaggaaaag 3420 aaagatgaac tctgcaggct aagaaatgaa aaagaacaag agctggacac attaaaaaga 3480 aagagtccat cagatttgtg gaaagaagac ttggctacat ttattgaaga attggaggct 3540 gttgaagcca aggaaaaaca agatgaacaa gtcggacttc ctgggaaagg ggggaaggcc 3600 aaggggaaaa aaacacaaat ggctgaagtt ttgccttctc cgcgtggtca aagagtcatt 3660 ccacgaataa ccatagaaat gaaagcagag gcagaaaaga aaaataaaaa gaaaattaag 3720 aatgaaaata ctgaaggaag ccctcaagaa gatggtgtgg aactagaagg cctaaaacaa 3780 agattagaaa agaaacagaa aagagaacca ggtacaaaga caaagaaaca aactacattg 3840 gcatttaagc caatcaaaaa aggaaagaag agaaatccct ggtctgattc agaatcagat 3900 aggagcagtg acgaaagtaa ttttgatgtc cctccacgag aaacagagcc acggagagca 3960 gcaacaaaaa caaaattcac aatggatttg gattcagatg aagatttctc agattttgat 4020 gaaaaaactg atgatgaaga ttttgtccca tcagatgcta gtccacctaa gaccaaaact 4080 tccccaaaac ttagtaacaa agaactgaaa ccacagaaaa gtgtcgtgtc agaccttgaa 4140 gctgatgatg ttaagggcag tgtaccactg tcttcaagcc ctcctgctac acatttccca 4200 gatgaaactg aaattacaaa cccagttcct aaaaagaatg tgacagtgaa gaagacagca 4260 gcaaaaagtc agtcttccac ctccactacc ggtgccaaaa aaagggctgc cccaaaagga 4320 actaaaaggg atccagcttt gaattctggt gtctctcaaa agcctgatcc tgccaaaacc 4380 aagaatcgcc gcaaaaggaa gccatccact tctgatgatt ctgactctaa ttttgagaaa 4440 attgtttcga aagcagtcac aagcaagaaa tccaaggggg agagtgatga cttccatatg 4500 gactttgact cagctgtggc tcctcgggca aaatctgtac gggcaaagaa acctataaag 4560 tacctggaag agtcagatga agatgatctg ttttaa 4596 <210> 15 <211> 1578 <212> DNA <213> Artificial Sequence <220> <223> PRC1 cDNA polynucleotide sequence <400> 15 atgaggagaa gtgaggtgct ggcggaggag tccatagtat gtctgcagaa agccctaaat 60 caccttcggg aaatatggga gctaattggg attccagagg accagcggtt acaaagaact 120 gaggtggtaa agaagcatat caaggaagaa ggagagacga ccatcttgca actagaaaaa 180 gatttgcgca cccaagtgga attgatgcga aaacagaaaa aggagagaaa acaggaactg 240 aagctacttc aagagcaaga tcaagaactg tgcgaaattc tttgtatgcc ccactatgat 300 attgacagtg cctcagtgcc cagcttagaa gagctgaacc agttcaggca acatgtgaca 360 actttgaggg aaacaaaggc ttctaggcgt gaggagtttg tcagtataaa gagacagatc 420 atactgtgta tggaagcatt agaccacacc ccagacacaa gctttgaaag agatgtggtg 480 tgtgaagacg aagatgcctt ttgtttgtct ttggagaata ttgcaacact acaaaagttg 540 ctacggcagc tggaaatgca gaaatcacaa aatgaagcag tgtgtgaggg gctgcgtact 600 caaatccgag agctctggga caggttgcaa atacctgaag aagaaagaga agctgtggcc 660 accattatgt ctgggtcaaa ggccaaggtc cggaaagcgc tgcaattaga agtggatcgg 720 ttggaagaac tgaaaatgca aaacatgaag aaagtgattg aggcaattcg agtggagctg 780 gttcagtact gggaccagtg cttttatagc caggagcaga gacaagcttt tgcccctttc 840 tgtgctgagg actacacaga aagtctgctc cagctccacg atgctgagat tgtgcggtta 900 aaaaactact atgaagttca caaggaactc tttgaaggtg tccagaagtg ggaagaaacc 960 tggaggcttt tcttagagtt tgagagaaaa gcttcagatc caaatcgatt tacaaaccga 1020 ggaggaaatc ttctaaaaga agaaaaacaa cgagccaagc tccagaaaat gctgcccaag 1080 ctggaagaag agttgaaggc acgaattgaa ttgtgggaac aggaacattc aaaggcattt 1140 atggtgaatg ggcagaaatt catggagtat gtggcagaac aatgggagat gcatcgattg 1200 gagaaagaga gagccaagca ggaaagacaa ctgaagaaca aaaaacagac agagacagag 1260 atgctgtatg gcagcgctcc tcgaacacct agcaagcggc gaggactggc tcccaataca 1320 ccgggcaaag cacgtaagct gaacactacc accatgtcca atgctacggc caatagtagc 1380 attcggccta tctttggagg gacagtctac cactcccccg tgtctcgact tcctccttct 1440 ggcagcaagc cagtcgctgc ttccacctgt tcagggaaga aaacaccccg tactggcagg 1500 catggagcca acaaggagaa cctggagctc aacggcagca tcctgagtgc gagaactttc 1560 aaaggcttcc aaatctga 1578 <210> 16 <211> 2517 <212> DNA <213> Artificial Sequence <220> <223> TACC3 cDNA polynucleotide sequence <400> 16 atgagtctgc aggtcttaaa cgacaaaaat gtcagcaatg aaaaaaatac agaaaattgc 60 gacttcctgt tttcgccacc agaagttacc ggaagatcgt ctgttcttcg tgtgtcacag 120 aaagaaaatg tgccacccaa gaacctggcc aaagctatga aggtgacttt tcagacacct 180 ctgcgggatc cacagacgca caggattcta agtcctagca tggccagcaa acttgaggct 240 cctttcactc aggatgacac ccttggactg gaaaactcac acccggtctg gacacagaaa 300 gagaaccaac agctcatcaa ggaagtggat gccaaaacta ctcatggaat tctacagaaa 360 ccagtggagg ctgacaccga cctcctgggg gatgcaagcc cagcctttgg gagtggcagc 420 tccagcgagt ctggcccagg tgccctggct gacctggact gctcaagctc ttcccagagc 480 ccaggaagtt ctgagaacca aatggtgtct ccaggaaaag tgtctggcag ccctgagcaa 540 gccgtggagg aaaaccttag ttcctattcc ttagacagaa gagtgacacc cgcctctgag 600 accctagaag acccttgcag gacagagtcc cagcacaaag cggagactcc gcacggagcc 660 gaggaagaat gcaaagcgga gactccgcac ggagccgagg aggaatgccg gcacggtggg 720 gtctgtgctc ccgcagcagt ggccacttcg cctcctggtg caatccctaa ggaagcctgc 780 ggaggagcac ccctgcaggg tctgcctggc gaagccctgg gctgccctgc gggtgtgggc 840 acccccgtgc cagcagatgg cactcagacc cttacctgtg cacacacctc tgctcctgag 900 agcacagccc caaccaacca cctggtggct ggcagggcca tgaccctgag tcctcaggaa 960 gaagtggctg caggccaaat ggccagctcc tcgaggagcg gacctgtaaa actagaattt 1020 gatgtatctg atggcgccac cagcaaaagg gcacccccac caaggagact gggagagagg 1080 tccggcctca agcctccctt gaggaaagca gcagtgaggc agcaaaaggc cccgcaggag 1140 gtggaggagg acgacggtag gagcggagca ggagaggacc cccccatgcc agcttctcgg 1200 ggctcttacc acctcgactg ggacaaaatg gatgacccaa acttcatccc gttcggaggt 1260 gacaccaagt ctggttgcag tgaggcccag cccccagaaa gccctgagac caggctgggc 1320 cagccagcgg ctgaacagtt gcatgctggg cctgccacgg aggagccagg tccctgtctg 1380 agccagcagc tgcattcagc ctcagcggag gacacgcctg tggtgcagtt ggcagccgag 1440 accccaacag cagagagcaa ggagagagcc ttgaactctg ccagcacctc gcttcccaca 1500 agctgtccag gcagtgagcc agtgcccacc catcagcagg ggcagcctgc cttggagctg 1560 aaagaggaga gcttcagaga ccccgctgag gttctaggca cgggcgcgga ggtggattac 1620 ctggagcagt ttggaacttc ctcgtttaag gagtcggcct tgaggaagca gtccttatac 1680 ctcaagttcg accccctcct gagggacagt cctggtagac cagtgcccgt ggccaccgag 1740 accagcagca tgcacggtgc aaatgagact ccctcaggac gtccgcggga agccaagctt 1800 gtggagttcg atttcttggg agcactggac attcctgtgc caggcccacc cccaggtgtt 1860 cccgcgcctg ggggcccacc cctgtccacc ggacctatag tggacctgct ccagtacagc 1920 cagaaggacc tggatgcagt ggtaaaggcg acacaggagg agaaccggga gctgaggagc 1980 aggtgtgagg agctccacgg gaagaacctg gaactgggga agatcatgga caggttcgaa 2040 gaggttgtgt accaggccat ggaggaagtt cagaagcaga aggaactttc caaagctgaa 2100 atccagaaag ttctaaaaga aaaagaccaa cttaccacag atctgaactc catggagaag 2160 tccttctccg acctcttcaa gcgttttgag aaacagaaag aggtgatcga gggctaccgc 2220 aagaacgaag agtcactgaa gaagtgcgtg gaggattacc tggcaaggat cacccaggag 2280 ggccagaggt accaagccct gaaggcccac gcggaggaga agctgcagct ggcaaacgag 2340 gagatcgccc aggtccggag caaggcccag gcggaagcgt tggccctcca ggccagcctg 2400 aggaaggagc agatgcgcat ccagtcgctg gagaagacag tggagcagaa gactaaagag 2460 aacgaggagc tgaccaggat ctgcgacgac ctcatctcca agatggagaa gatctga 2517 <210> 17 <211> 2715 <212> DNA <213> Artificial Sequence <220> <223> MCM2 cDNA polynucleotide sequence <400> 17 atggcggaat catcggaatc cttcaccatg gcatccagcc cggcccagcg tcggcgaggc 60 aatgatcctc tcacctccag ccctggccga agctcccggc gtactgatgc cctcacctcc 120 agccctggcc gtgaccttcc accatttgag gatgagtccg aggggctcct aggcacagag 180 gggcccctgg aggaagaaga ggatggagag gagctcattg gagatggcat ggaaagggac 240 taccgcgcca tcccagagct ggacgcctat gaggccgagg gactggctct ggatgatgag 300 gacgtagagg agctgacggc cagtcagagg gaggcagcag agcgggccat gcggcagcgt 360 gaccgggagg ctggccgggg cctgggccgc atgcgccgtg ggctcctgta tgacagcgat 420 gaggaggacg aggagcgccc tgcccgcaag cgccgccagg tggagcgggc cacggaggac 480 ggcgaggagg acgaggagat gatcgagagc atcgagaacc tggaggatct caaaggccac 540 tctgtgcgcg agtgggtgag catggcgggc ccccggctgg agatccacca ccgcttcaag 600 aacttcctgc gcactcacgt cgacagccac ggccacaacg tcttcaagga gcgcatcagc 660 gacatgtgca aagagaaccg tgagagcctg gtggtgaact atgaggactt ggcagccagg 720 gagcacgtgc tggcctactt cctgcctgag gcaccggcgg agctgctgca gatctttgat 780 gaggctgccc tggaggtggt actggccatg taccccaagt acgaccgcat caccaaccac 840 atccatgtcc gcatctccca cctgcctctg gtggaggagc tgcgctcgct gaggcagctg 900 catctgaacc agctgatccg caccagtggg gtggtgacca gctgcactgg cgtcctgccc 960 cagctcagca tggtcaagta caactgcaac aagtgcaatt tcgtcctggg tcctttctgc 1020 cagtcccaga accaggaggt gaaaccaggc tcctgtcctg agtgccagtc ggccggcccc 1080 tttgaggtca acatggagga gaccatctat cagaactacc agcgtatccg aatccaggag 1140 agtccaggca aagtggcggc tggccggctg ccccgctcca aggacgccat tctcctcgca 1200 gatctggtgg acagctgcaa gccaggagac gagatagagc tgactggcat ctatcacaac 1260 aactatgatg gctccctcaa cactgccaat ggcttccctg tctttgccac tgtcatccta 1320 gccaaccacg tggccaagaa ggacaacaag gttgctgtag gggaactgac cgatgaagat 1380 gtgaagatga tcactagcct ctccaaggat cagcagatcg gagagaagat ctttgccagc 1440 attgctcctt ccatctatgg tcatgaagac atcaagagag gcctggctct ggccctgttc 1500 ggaggggagc ccaaaaaccc aggtggcaag cacaaggtac gtggtgatat caacgtgctc 1560 ttgtgcggag accctggcac agcgaagtcg cagtttctca agtatattga gaaagtgtcc 1620 agccgagcca tcttcaccac tggccagggg gcgtcggctg tgggcctcac ggcgtatgtc 1680 cagcggcacc ctgtcagcag ggagtggacc ttggaggctg gggccctggt tctggctgac 1740 cgaggagtgt gtctcattga tgaatttgac aagatgaatg accaggacag aaccagcatc 1800 catgaggcca tggagcaaca gagcatctcc atctcgaagg ctggcatcgt cacctccctg 1860 caggctcgct gcacggtcat tgctgccgcc aaccccatag gagggcgcta cgacccctcg 1920 ctgactttct ctgagaacgt ggacctcaca gagcccatca tctcacgctt tgacatcctg 1980 tgtgtggtga gggacaccgt ggacccagtc caggacgaga tgctggcccg cttcgtggtg 2040 ggcagccacg tcagacacca ccccagcaac aaggaggagg aggggctggc caatggcagc 2100 gctgctgagc ccgccatgcc caacacgtat ggcgtggagc ccctgcccca ggaggtcctg 2160 aagaagtaca tcatctacgc caaggagagg gtccacccga agctcaacca gatggaccag 2220 gacaaggtgg ccaagatgta cagtgacctg aggaaagaat ctatggcgac aggcagcatc 2280 cccattacgg tgcggcacat cgagtccatg atccgcatgg cggaggccca cgcgcgcatc 2340 catctgcggg actatgtgat cgaagacgac gtcaacatgg ccatccgcgt gatgctggag 2400 agcttcatag acacacagaa gttcagcgtc atgcgcagca tgcgcaagac ttttgcccgc 2460 tacctttcat tccggcgtga caacaatgag ctgttgctct tcatactgaa gcagttagtg 2520 gcagagcagg tgacatatca gcgcaaccgc tttggggccc agcaggacac tattgaggtc 2580 cctgagaagg acttggtgga taaggctcgt cagatcaaca tccacaacct ctctgcattt 2640 tatgacagtg agctcttcag gatgaacaag ttcagccacg acctgaaaag gaaaatgatc 2700 ctgcagcagt tctga 2715 <210> 18 <211> 3627 <212> DNA <213> Artificial Sequence <220> <223> RECQL4 cDNA polynucleotide sequence <400> 18 atggagcggc tgcgggacgt gcgggagcgg ctgcaggcgt gggagcgcgc gttccgacgg 60 cagcgcgggc ggcgaccgag ccaggacgac gtggaggcgg cgccggagga gacccgcgcg 120 ctctaccggg aataccgcac tctgaagcgt accacgggcc aggccggcgg cgggctccgc 180 agctccgagt cgctccccgc ggcggccgaa gaggcgccag agccccgctg ctgggggccc 240 catctgaatc gggctgcgac caagagtcca cagtctacgc cagggcggag ccgccagggc 300 tcggtgccgg actacgggca gcggctcaag gccaatctga aaggcaccct gcaggccgga 360 ccagccctgg gccgcagacc gtggcctcta ggaagagcct catctaaggc atccacccca 420 aagcccccag gtacagggcc tgtcccctcc tttgcagaaa aagtcagtga tgagcctcca 480 cagctccctg agccccagcc aaggccaggc cggctccagc atctgcaggc atccctgagc 540 cagcggctgg gctccctaga tcctggctgg ttacagcgat gtcacagtga ggtcccagat 600 tttctggggg cccccaaagc ctgcaggcct gatctaggct cagaggaatc acaacttctg 660 atccctggtg agtcggctgt ccttggtcct ggtgctggct cccagggccc agaggcttca 720 gccttccaag aagtcagcat ccgtgtgggg agcccccagc ccagcagcag tggaggcgag 780 aagcggagat ggaacgagga gccctgggag agccccgcac aggtccagca ggagagcagc 840 caagctggac ccccatcgga gggggctggg gctgtagcag ttgaggaaga ccctccaggg 900 gaacctgtac aggcacagcc acctcagccc tgcagcagcc catcgaaccc caggtaccac 960 ggactcagcc cctccagtca agctagggct gggaaggctg agggcacagc ccccctgcac 1020 atcttccctc ggctggcccg ccatgacagg ggcaattacg tacggctcaa catgaagcag 1080 aaacactacg tgcggggccg ggcactccgt agcaggctcc tccgcaagca ggcatggaag 1140 cagaagtggc ggaagaaagg ggagtgtttt gggggtggtg gtgccacagt cacaaccaag 1200 gagtcttgtt tcctgaacga gcagttcgat cactgggcag cccagtgtcc ccggccagca 1260 agtgaggaag acacagatgc tgttgggcct gagccactgg ttccttcacc acaacctgta 1320 cctgaggtgc ccagcctgga ccccaccgtg ctgccactct actccctggg gccctcaggg 1380 cagttggcag agacgccggc tgaggtgttc caggccctgg agcagctggg gcaccaagcc 1440 tttcgccctg ggcaggagcg tgcagtcatg cggatcctgt ctggcatctc cacgctgctg 1500 gtgctgccta caggtgccgg caagtccctg tgctaccagc tcccagcgct gctctacagc 1560 cggcgcagcc cctgcctcac gttggtcgtc tctcccctgc tgtcactcat ggatgaccag 1620 gtgtctggcc tgccaccgtg tctcaaggcg gcctgcatac actcgggcat gaccaggaag 1680 caacgggaat ctgtcctgca gaagattcgg gcagcccagg tacacgtgct gatgctgaca 1740 cctgaggcac tggtgggggc gggaggcctc cctccagccg cacagctgcc tccagttgct 1800 tttgcctgca ttgatgaggc ccactgcctc tcccagtggt cccacaactt ccggccctgc 1860 tacctgcgcg tctgcaaggt gcttcgggag cgcatgggcg tgcactgctt cctgggcctc 1920 acagccacag ccacacgccg cactgccagt gacgtggcac agcacctggc tgtggctgaa 1980 gagcctgacc tccacgggcc agccccagtt cccaccaacc tgcacctttc cgtgtccatg 2040 gacagggaca cagaccaggc actgttgacg ctgctgcaag gcaaacgttt tcaaaacctc 2100 gattccatta tcatttactg caaccggcgc gaggacacag agcggatcgc tgcgctcctc 2160 cgaacctgcc tgcacgcagc ctgggtccca gggtctggag gtcgtgcccc caaaaccaca 2220 gccgaggcct accacgcggg catgtgcagc cgggaacggc ggcgggtaca gcgagccttc 2280 atgcagggcc agttgcgggt ggtggtggcc acggtggcct ttgggatggg gctggaccgg 2340 ccagatgtgc gggctgtgct gcatctgggg ctgcccccaa gcttcgagag ctacgtgcag 2400 gccgtgggcc gggccgggcg tgacgggcag cctgcccact gccacctctt cctgcagccc 2460 cagggcgaag acctgcgaga gctgcgcaga catgtgcacg ccgacagcac ggacttcctg 2520 gctgtgaaga ggctggtaca gcgcgtgttc ccagcctgca cctgcacctg caccaggccg 2580 ccctcggagc aggaaggggc cgtgggtggg gagaggcctg tgcccaagta cccccctcaa 2640 gaggctgagc agcttagcca ccaagcagcc ccaggaccca gaagggtctg catgggccat 2700 gagcgggcac tcccaataca gcttaccgta caggctttgg acatgccgga ggaggccatc 2760 gagactttgc tgtgctacct ggagctgcac ccacaccact ggctggagct gctggcgacc 2820 acctataccc attgccgtct gaactgccct gggggccctg cccagctcca ggccctggcc 2880 cacaggtgtc cccctttggc tgtgtgcttg gcccagcagc tgcctgagga cccagggcaa 2940 ggcagcagct ccgtggagtt tgacatggtc aagctggtgg actccatggg ctgggagctg 3000 gcctctgtgc ggcgggctct ctgccagctg cagtgggacc acgagcccag gacaggtgtg 3060 cggcgtggga caggggtgct tgtggagttc agtgagctgg ccttccacct tcgcagcccg 3120 ggggacttga ccgctgagga gaaggaccag atatgtgact tcctctatgg ccgtgtgcag 3180 gcccgggagc gccaggccct ggcccgtctg cgcagaacct tccaggcctt tcacagcgta 3240 gccttcccca gctgcgggcc ctgcctggag cagcaggatg aggagcgcag caccaggctc 3300 aaggacctgc tcggccgcta ctttgaggaa gaggaagggc aggagccggg aggcatggag 3360 gacgcacagg gccccgagcc agggcaggcc agactccagg attgggagga ccaggtccgc 3420 tgcgacatcc gccagttcct gtccctgagg ccagaggaga agttctccag cagggctgtg 3480 gcccgcatct tccacggcat cggaagcccc tgctacccgg cccaggtgta cgggcaggac 3540 cgacgcttct ggagaaaata cctgcacctg agcttccatg ccctggtggg cctggccacg 3600 gaagagctcc tgcaggtggc ccgctga 3627 <210> 19 <211> 3582 <212> DNA <213> Artificial Sequence <220> <223> SPAG5 cDNA polynucleotide sequence <400> 19 atgtggcgag tgaaaaaact gagcctcagc ctgtcgcctt cgccccagac gggaaaacca 60 tctatgagaa ctcctctccg tgaacttacc ctgcagcccg gtgccctcac caactctgga 120 aaaagatccc ccgcttgctc ctcgctgacc ccatcactgt gcaagctggg gctgcaggaa 180 ggcagcaaca actcatctcc agtggatttt gtaaataaca agaggacaga cttatcttca 240 gaacatttca gtcattcctc aaagtggcta gaaacttgtc agcatgaatc agatgagcag 300 cctctagatc caattcccca aattagctct actcctaaaa cgtctgagga agcagtagac 360 ccactgggca attatatggt taaaaccatc gtccttgtac catctccact ggggcagcaa 420 caagacatga tatttgaggc ccgtttagat accatggcag agacaaacag catatcttta 480 aatggacctt tgagaacaga cgatctggtg agagaggagg tggcaccctg catgggagac 540 aggttttcag aagttgctgc tgtatctgag aaacctatct ttcaggaatc tccgtcccat 600 ctcttagagg agtctccacc aaatccctgt tctgaacaac tacattgctc caaggaaagc 660 ctgagcagta gaactgaggc tgtgcgtgag gacttagtac cttctgaaag taacgccttc 720 ttgccttcct ctgttctctg gctttcccct tcaactgcct tggcagcaga tttccgtgtc 780 aatcatgtgg acccagagga ggaaattgta gagcatggag ctatggagga aagagaaatg 840 aggtttccca cacatcctaa ggagtctgaa acagaagatc aagcacttgt ctcaagtgtg 900 gaagatattc tgtccacatg cctgacacca aatctagtag aaatggaatc ccaagaagct 960 ccaggcccag cagtagaaga tgttggtagg attcttggct ctgatacaga gtcttggatg 1020 tccccactgg cctggctgga aaaaggtgta aatacctccg tcatgctgga aaatctccgc 1080 caaagcttat cccttccctc gatgcttcgg gatgctgcaa ttggcactac ccctttctct 1140 acttgctcgg tggggacttg gtttactcct tcagcaccac aggaaaagag tacaaacaca 1200 tcccagacag gcctggttgg caccaagcac agtacttctg agacagagca gctcctgtgt 1260 ggccggcctc cagatctgac tgccttgtct cgacatgact tggaagataa cctgctgagc 1320 tctcttgtca ttctggaggt tctctcccgc cagcttcggg actggaagag ccagctggct 1380 gtccctcacc cagaaaccca ggacagtagc acacagactg acacatctca cagtgggata 1440 actaataaac ttcagcatct taaggagagc catgagatgg gacaggccct acagcaggcc 1500 agaaatgtca tgcaatcatg ggtgcttatc tctaaagagc tgatatcctt gcttcaccta 1560 tccctgttgc atttagaaga agataagact actgtgagtc aggagtctcg gcgtgcagaa 1620 acattggtct gttgctgttt tgatttgctg aagaaattga gggcaaagct ccagagcctc 1680 aaagcagaaa gggaggaggc aaggcacaga gaggaaatgg ctctcagagg caaggatgcg 1740 gcagagatag tgttggaggc tttctgtgca cacgccagcc agcgcatcag ccagctggaa 1800 caggacctag catccatgcg ggaattcaga ggccttctga aggatgccca gacccaactg 1860 gtagggcttc atgccaagca agaagagctg gttcagcaga cagtgagtct tacttctacc 1920 ttgcaacaag actggaggtc catgcaactg gattatacaa catggacagc tttgctgagt 1980 cggtcccgac aactcacaga gaaactcaca gtcaagagcc agcaagccct gcaggaacgt 2040 gatgtggcaa ttgaggaaaa gcaggaggtt tctagggtgc tggaacaagt ctctgcccag 2100 ttagaggagt gcaaaggcca aacagaacaa ctggagttgg aaaacagtcg tctagcaaca 2160 gatctccggg ctcagttgca gattctggcc aacatggaca gccagctaaa agagctacag 2220 agtcagcata cccattgtgc ccaggacctg gctatgaagg atgagttact ctgccagctt 2280 acccagagca atgaggagca ggctgctcaa tggcaaaagg aagagatggc actaaaacac 2340 atgcaggcag aactgcagca gcaacaagct gtcctggcca aagaggtgcg ggacctgaaa 2400 gagaccttgg agtttgcaga ccaggagaat caggttgctc acctggagct gggtcaggtt 2460 gagtgtcaat tgaaaaccac actggaagtg ctccgggagc gcagcttgca gtgtgagaac 2520 ctcaaggaca ctgtagagaa cctaacggct aaactggcca gcaccatagc agataaccag 2580 gagcaagatc tggagaaaac acggcagtac tctcaaaagc tagggctgct gactgagcaa 2640 ctacagagcc tgactctctt tctacagaca aaactaaagg agaagactga acaagagacc 2700 cttctgctga gtacagcctg tcctcccacc caggaacacc ctctgcctaa tgacaggacc 2760 ttcctgggaa gcatcttgac agcagtggca gatgaagagc cagaatcaac tcctgtgccc 2820 ttgcttggaa gtgacaagag tgctttcacc cgagtagcat caatggtttc ccttcagccc 2880 gcagagaccc caggcatgga ggagagcctg gcagaaatga gtattatgac tactgagctt 2940 cagagtcttt gttccctgct acaagagtct aaagaagaag ccatcaggac tctgcagcga 3000 aaaatttgtg agctgcaagc taggctgcag gcccaggaag aacagcatca ggaagtccag 3060 aaggcaaaag aagcagacat agagaagctg aaccaggcct tgtgcttgcg ctacaagaat 3120 gaaaaggagc tccaggaagt gatacagcag cagaatgaga agatcctaga acagatagac 3180 aagagtggcg agctcataag ccttagagag gaggtgaccc accttacccg ctcacttcgg 3240 cgtgcggaga cagagaccaa agtgctccag gaggccctgg caggccagct ggactccaac 3300 tgccagccta tggccaccaa ttggatccag gagaaagtgt ggctctctca ggaggtggac 3360 aaactgagag tgatgttcct ggagatgaaa aatgagaagg aaaaactcat gatcaagttc 3420 cagagccata gaaatatcct agaggagaac cttcggcgct ctgacaagga gttagaaaaa 3480 ctagatgaca ttgttcagca tatttataag accctgctct ctattccaga ggtggtgagg 3540 ggatgcaaag aactacaggg attgctggaa tttctgagct aa 3582 <110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Use of BUB1 as a biomarker for predicting the prognosis of Non-muscle invasive bladder cancer <130> NPDC-96617 <160> 19 <170> KoPatentIn 3.0 <210> 1 <211> 3198 <212> DNA <213> Artificial Sequence <220> <223> BUB1 cDNA polynucleotide sequence <400> 1 atggacaccc cggaaaatgt ccttcaatac atacagtggg tagaagagaa ttttcctgag 60 aataaagaat acttgataac tttactagaa catttaatga aggaattttt agataagaag 120 aaataccaca atgacccaag attcatcagt tattgtttaa aatttgctga gtacaacagt 180 gacctccatc aattttttga gtttctgtac aaccatggga ttggaaccct gtcatcccct 240 ctgtacattg cctgggcggg gcatctggaa gcccaaggag agctgcagca tgccagtgct 300 gtccttcaga gaggaattca aaaccaggct gaacccagag agttcctgca acaacaatac 360 aggttatttc agacacgcct cactgaaacc catttgccag ctcaagctag aacctcagaa 420 cctctgcata atgttcaggt tttaaatcaa atgataacat caaaatcaaa tccaggaaat 480 aacatggcct gcatttctaa gaatcagggt tcagagcttt ctggagtgat atcttcagct 540 tgtgataaag agtcaaatat ggaacgaaga gtgatc acga tttctaaatc agaatattct 600 gtgcactcat ctttggcatc caaagttgat gttgagcagg ttgttatgta ttgcaaggag 660 aagcttattc gtggggaatc agaattttcc tttgaagaat tgagagccca gaaatacaat 720 caacggagaa agcatgagca atgggtaaat gaagacagac attatatgaa aaggaaagaa 780 gcaaatgctt ttgaagaaca gctattaaaa cagaaaatgg atgaacttca taagaagttg 840 catcaggtgg tggagacatc ccatgaggat ctgcccgctt cccaggaaag gtccgaggtt 900 aatccagcac gtatggggcc aagtgtaggc tcccagcagg aactgagagc gccatgtctt 960 ccagtaacct atcagcagac accagtgaac atggaaaaga acccaagaga ggcacctcct 1020 gttgttcctc ctttggcaaa tgctatttct gcagctttgg tgtccccagc caccagccag 1080 agcattgctc ctcctgttcc tttgaaagcc cagacagtaa cagactccat gtttgcagtg 1140 gccagcaaag atgctggatg tgtgaataag agtactcatg aattcaagcc acagagtgga 1200 gcagagatca aagaagggtg tgaaacacat aaggttgcca acacaagttc ttttcacaca 1260 actccaaaca catcactggg aatggttcag gcaacgccat ccaaagtgca gccatcaccc 1320 accgtgcaca caaaagaagc attaggtttc atcatgaata tgtttcaggc tcctacactt 1380 cctgatattt ctgatgacaa agatgaatgg caatctctag atcaaaat ga agatgcattt 1440 gaagcccagt ttcaaaaaaa tgtaaggtca tctggggctt ggggagtcaa taagatcatc 1500 tcttctttgt catctgcttt tcatgtgttt gaagatggaa acaaagaaaa ttatggatta 1560 ccacagccta aaaataaacc cacaggagcc aggacctttg gagaacgctc tgtcagcaga 1620 cttccttcaa aaccaaagga ggaagtgcct catgctgaag agtttttgga tgactcaact 1680 gtatggggta ttcgctgcaa caaaaccctg gcacccagtc ctaagagccc aggagacttc 1740 acatctgctg cacaacttgc gtctacacca ttccacaagc ttccagtgga gtcagtgcac 1800 attttagaag ataaagaaaa tgtggtagca aaacagtgta cccaggcgac tttggattct 1860 tgtgaggaaa acatggtggt gccttcaagg gatggaaaat tcagtccaat tcaagagaaa 1920 agcccaaaac aggccttgtc gtctcacatg tattcagcat ccttacttcg tctgagccag 1980 cctgctgcag gtggggtact tacctgtgag gcagagttgg gcgttgaggc ttgcagactc 2040 acagacactg acgctgccat tgcagaagat ccaccagatg ctattgctgg gctccaagca 2100 gaatggatgc agatgagttc acttgggact gttgatgctc caaacttcat tgttgggaac 2160 ccatgggatg ataagctgat tttcaaactt ttatctgggc tttctaaacc agtgagttcc 2220 tatccaaata cttttgaatg gcaatgtaaa cttccagcca tcaagcccaa gac tgaattt 2280 caattgggtt ctaagctggt ctatgtccat caccttcttg gagaaggagc ctttgcccag 2340 gtgtacgaag ctacccaggg agatctgaat gatgctaaaa ataaacagaa atttgtttta 2400 aaggtccaaa agcctgccaa cccctgggaa ttctacattg ggacccagtt gatggaaaga 2460 ctaaagccat ctatgcagca catgtttatg aagttctatt ctgcccactt attccagaat 2520 ggcagtgtat tagtaggaga gctctacagc tatggaacat tattaaatgc cattaacctc 2580 tataaaaata cccctgaaaa agtgatgcct caaggtcttg tcatctcttt tgctatgaga 2640 atgctttaca tgattgagca agtgcatgac tgtgaaatca ttcatggaga cattaaacca 2700 gacaatttca tacttggaaa cggatttttg gaacaggatg atgaagatga tttatctgct 2760 ggcttggcac tgattgacct gggtcagagt atagatatga aactttttcc aaaaggaact 2820 atattcacag caaagtgtga aacatctggt tttcagtgtg ttgagatgct cagcaacaaa 2880 ccatggaact accagatcga ttactttggg gttgctgcaa cagtatattg catgctcttt 2940 ggcacttaca tgaaagtgaa aaatgaagga ggagagtgta agcctgaagg tctttttaga 3000 aggcttcctc atttggatat gtggaatgaa ttttttcatg ttatgttgaa tattccagat 3060 tgtcatcatc ttccatcttt ggatttgtta aggcaaaagc tgaagaaagt atttcaaca a 3120 cactatacta acaagattag ggccctacgt aataggctaa ttgtactgct cttagaatgt 3180 aagcgttcac gaaaataa 3198 <210> 2 <211> 2382 <212> DNA <213> Artificial Sequence <220> <223> UHRF1 cDNA polynucleotide sequence <400> 2 atgtggatcc aggttcggac catggacggg aggcagaccc acacggtgga ctcgctgtcc 60 aggctgacca aggtggagga gctgaggcgg aagatccagg agctgttcca cgtggagcca 120 ggcctgcaga ggctgttcta caggggcaaa cagatggagg acggccatac cctcttcgac 180 tacgaggtcc gcctgaatga caccatccag ctcctggtcc gccagagcct cgtgctcccc 240 cacagcacca aggagcggga ctccgagctc tccgacaccg actccggctg ctgcctgggc 300 cagagtgagt cagacaagtc ctccacccac ggtgaggcgg ccgccgagac tgacagcagg 360 ccagccgatg aggacatgtg ggatgagacg gaattggggc tgtacaaggt caatgagtac 420 gtcgatgctc gggacacgaa catgggggcg tggtttgagg cgcaggtggt cagggtgacg 480 cggaaggccc cctcccggga cgagccctgc agctccacgt ccaggccggc gctggaggag 540 gacgtcattt accacgtgaa atacgacgac tacccggaga acggcgtggt ccagatgaac 600 tccagggacg tccgagcgcg cgcccgcacc atcatcaagt ggcaggacct ggaggtgggc 660 caggtcaggtca cta caaccccgac aaccccaagg agcggggctt ctggtacgac 720 gcggagatct ccaggaagcg cgagaccagg acggcgcggg aactctacgc caacgtggtg 780 ctgggggatg attctctgaa cgactgtcgg atcatcttcg tggacgaagt cttcaagatt 840 gagcggccgg gtgaagggag ccccatggtt gacaacccca tgagacggaa gagcgggccg 900 tcctgcaagc actgcaagga cgacgtgaac agactctgcc gggtctgcgc ctgccacctg 960 tgcgggggcc ggcaggaccc cgacaagcag ctcatgtgcg atgagtgcga catggccttc 1020 cacatctact gcctggaccc gcccctcagc agtgttccca gcgaggacga gtggtactgc 1080 cctgagtgcc ggaatgatgc cagcgaggtg gtactggcgg gagagcggct gagagagagc 1140 aagaagaagg cgaagatggc ctcggccaca tcgtcctcac agcgggactg gggcaagggc 1200 atggcctgtg tgggccgcac caaggaatgt accatcgtcc cgtccaacca ctacggaccc 1260 atcccgggga tccccgtggg caccatgtgg cggttccgag tccaggtcag cgagtcgggt 1320 gtccatcggc cccacgtggc tggcatacac ggccggagca acgacggagc gtactcccta 1380 gtcctggcgg ggggctatga ggatgatgtg gaccatggga attttttcac atacacgggt 1440 agtggtggtc gagatctttc cggcaacaag aggaccgcgg aacagtcttg tgatcagaaa 1500 ctcaccaaca ccaacagggc gctggct ctc aactgctttg ctcccatcaa tgaccaagaa 1560 ggggccgagg ccaaggactg gcggtcgggg aagccggtca gggtggtgcg caatgtcaag 1620 ggtggcaaga atagcaagta cgcccccgct gagggcaacc gctacgatgg catctacaag 1680 gttgtgaaat actggcccga gaaggggaag tccgggtttc tcgtgtggcg ctaccttctg 1740 cggagggacg atgatgagcc tggcccttgg acgaaggagg ggaaggaccg gatcaagaag 1800 ctggggctga ccatgcagta tccagaaggc tacctggaag ccctggccaa ccgagagcga 1860 gagaaggaga acagcaagag ggaggaggag gagcagcagg aggggggctt cgcgtccccc 1920 aggacgggca agggcaagtg gaagcggaag tcggcaggag gtggcccgag cagggccggg 1980 tccccgcgcc ggacatccaa gaaaaccaag gtggagccct acagtctcac ggcccagcag 2040 agcagcctca tcagagagga caagagcaac gccaagctgt ggaatgaggt cctggcgtca 2100 ctcaaggacc ggccggcgag cggcagcccg ttccagttgt tcctgagtaa agtggaggag 2160 acgttccagt gtatctgctg tcaggagctg gtgttccggc ccatcacgac cgtgtgccag 2220 cacaacgtgt gcaaggactg cctggacaga tcctttcggg cacaggtgtt cagctgccct 2280 gcctgccgct acgacctggg ccgcagctat gccatgcagg tgaaccagcc tctgcagacc 2340 gtcctcaacc agctcttccc cggctacggc aa tggccggt ga 2382 <210> 3 <211> 912 <212> DNA <213> Artificial Sequence <220> <223> AURKB cDNA polynucleotide sequence <400> 3 atgagccgct ccaatgtcca gcccacagct gcccctggcc agaaggtgat ggagaatagc 60 agtgggacac ccgacatctt aacgcggcac ttcacaattg atgactttga gattgggcgt 120 cctctgggca aaggcaagtt tggaaacgtg tacttggctc gggagaagaa aagccatttc 180 atcgtggcgc tcaaggtcct cttcaagtcc cagatagaga aggagggcgt ggagcatcag 240 ctgcgcagag agatcgaaat ccaggcccac ctgcaccatc ccaacatcct gcgtctctac 300 aactattttt atgaccggag gaggatctac ttgattctag agtatgcccc ccgcggggag 360 ctctacaagg agctgcagaa gagctgcaca tttgacgagc agcgaacagc cacgatcatg 420 gaggagttgg cagatgctct aatgtactgc catgggaaga aggtgattca cagagacata 480 aagccagaaa atctgctctt agggctcaag ggagagctga agattgctga cttcggctgg 540 tctgtgcatg cgccctccct gaggaggaag acaatgtgtg gcaccctgga ctacctgccc 600 ccagagatga ttgaggggcg catgcacaat gagaaggtgg atctgtggtg cattggagtg 660 ctttgctatg agctgctggt ggggaaccca ccctttgaga gtgcatcaca caacgagacc 720 tatcgccgca tcgtcaaggt ggatcctaaaggt ccccgctt ccgtgcccat gggagcccag 780 gacctcatct ccaaactgct caggcataac ccctcggaac ggctgcccct ggcccaggtc 840 tcagcccacc cttgggtccg ggccaactct cggagggtgc tgcctccctc tgcccttcaa 900 tctgtcgcct ga 912 <210> 4 <211> 2242 <212> DNA <213> Artificial Sequence <220> <223> TPX2 cDNA polynucleotide sequence <400 > 4 atgatgaagg agatactcaa aacatagatt catggtttga ggagaaggcc aatttggaga 60 ataagttact ggggaagaat ggaactggag ggctttttca gggcaaaact cctttgagaa 120 aggctaatct tcagcaagct attgtcacac ctttgaaacc agttgacaac acttactaca 180 aagaggcaga aaaagaaaat cttgtggaac aatccattcc gtcaaatgct tgttcttccc 240 tggaagttga ggcagccata tcaagaaaaa ctccagccca gcctcagaga agatctctta 300 ggctttctgc tcagaaggat ttggaacaga aagaaaagca tcatgtaaaa atgaaagcca 360 agagatgtgc cactcctgta atcatcgatg aaattctacc ctctaagaaa atgaaagttt 420 ctaacaacaa aaagaagcca gaggaagaag gcagtgctca tcaagatact gctgaaaaga 480 atgcatcttc cccagagaaa gccaagggta gacatactgt gccttgtatg ccacctgcaa 540 agcagaagtt tctaaaaagt actgaggagc aagagctgga gaagagtatg aaaat000cagc aagaggtggt ggagatgcgg aaaaagaatg aagaattcaa gaaacttgct ctggctggaa 660 tagggcaacc tgtgaagaaa tcagtgagcc aggtcaccaa atcagttgac ttccacttcc 720 gcacagatga gcgaatcaaa caacatccta agaaccagga ggaatataag gaagtgaact 780 ttacatctga actacgaaag catccttcat ctcctgcccg agtgactaag ggatgtacca 840 ttgttaagcc tttcaacctg tcccaaggaa agaaaagaac atttgatgaa acagtttcta 900 catatgtgcc ccttgcacag caagttgaag acttccataa acgaacccct aacagatatc 960 atttgaggag caagaaggat gatattaacc tgttaccctc caaatcttct gtgaccaaga 1020 tttgcagaga cccacagact cctgtactgc aaaccaaaca ccgtgcacgg gctgtgacct 1080 gcaaaagtac agcagagctg gaggctgagg agctcgagaa attgcaacaa tacaaattca 1140 aagcacgtga acttgatccc agaatacttg aaggtgggcc catcttgccc aagaaaccac 1200 ctgtgaaacc acccaccgag cctattggct ttgatttgga aattgagaaa agaatccagg 1260 agcgagaatc aaagaagaaa acagaggatg aacactttga atttcattcc agaccttgcc 1320 ctactaagat tttggaagat gttgtgggtg ttcctgaaaa gaaggtactt ccaatcaccg 1380 tccccaagtc accagccttt gcattgaaga acagaattcg aatgcccacc aaagaagatg 1440 aggaagagga cgaaccggta gtgataaaag ctcaacctgt gccacattat ggggtgcctt 1500 ttaagcccca aatcccagag gcaagaactg tggaaatatg ccctttctcg tttgattctc 1560 gagacaaaga acgtcagtta cagaaggaga agaaaataaa agaactgcag aaaggggagg 1620 tgcccaagtt caaggcactt cccttgcctc attttgacac cattaacctg ccagagaaga 1680 aggtaaagaa tgtgacccag attgaacctt tctgcttgga gactgacaga agaggtgctc 1740 tgaaggcaca gacttggaag caccagctgg aagaagaact gagacagcag aaagaagcag 1800 cttgtttcaa ggctcgtcca aacaccgtca tctctcagga gccctttgtt cccaagaaag 1860 agaagaaatc agttgctgag ggcctttctg gttctctagt tcaggaacct tttcagctgg 1920 ctactgagaa gagagccaaa gagcggcagg agctggagaa gagaatggct gaggtagaag 1980 cccagaaagc ccagcagttg gaggaggcca gactacagga ggaagagcag aaaaaagagg 2040 agctggccag gctacggaga gaactggtgc ataaggcaaa tccaatacgc aagtaccagg 2100 gtctggagat aaagtcaagt gaccagcctc tgactgtgcc tgtatctccc aaattctcca 2160 ctcgattcca ctgctaaact cagctgtgag ctgcggatac cgcccggcaa tgggacctgc 2220 tcttaacctc aaacctagga cc 2242 <210> 5 <211 > 3160 <212> DNA <213> Artificial Sequence <22 0> <223> BUB1B cDNA polynucleotide sequence <400> 5 atgcaggatg gcggcggtga agaaggaagg gggtgctctg agtgaagcca tgtccctgga 60 gggagatgaa tgggaactga gtaaagaaaa tgtacaacct ttaaggcaag ggcggatcat 120 gtccacgctt cagggagcac tggcacaaga atctgcctgt aacaatactc ttcagcagca 180 gaaacgggca tttgaatatg aaattcgatt ttacactgga aatgaccctc tggatgtttg 240 ggataggtat atcagctgga cagagcagaa ctatcctcaa ggtgggaagg agagtaatat 300 gtcaacgtta ttagaaagag ctgtagaagc actacaagga gaaaaacgat attatagtga 360 tcctcgattt ctcaatctct ggcttaaatt agggcgttta tgcaatgagc ctttggatat 420 gtacagttac ttgcacaacc aagggattgg tgtttcactt gctcagttct atatctcatg 480 ggcagaagaa tatgaagcta gagaaaactt taggaaagca gatgcgatat ttcaggaagg 540 gattcaacag aaggctgaac cactagaaag actacagtcc cagcaccgac aattccaagc 600 tcgagtgtct cggcaaactc tgttggcact tgagaaagaa gaagaggagg aagtttttga 660 gtcttctgta ccacaacgaa gcacactagc tgaactaaag agcaaaggga aaaagacagc 720 aagagctcca atcatccgtg taggaggtgc tctcaaggct ccaagccaga acagaggact 780 ccaaaatcca tttcctcaac agatgcaaaa taatag taga attactgttt ttgatgaaaa 840 tgctgatgag gcttctacag cagagttgtc taagcctaca gtccagccat ggatagcacc 900 ccccatgccc agggccaaag agaatgagct gcaagcaggc ccttggaaca caggcaggtc 960 cttggaacac aggcctcgtg gcaatacagc ttcactgata gctgtacccg ctgtgcttcc 1020 cagtttcact ccatatgtgg aagagactgc acgacagcca gttatgacac catgtaaaat 1080 tgaacctagt ataaaccaca tcctaagcac cagaaagcct ggaaaggaag aaggagatcc 1140 tctacaaagg gttcagagcc atcagcaagc gtctgaggag aagaaagaga agatgatgta 1200 ttgtaaggag aagatttatg caggagtagg ggaattctcc tttgaagaaa ttcgggctga 1260 agttttccgg aagaaattaa aagagcaaag ggaagccgag ctattgacca gtgcagagaa 1320 gagagcagaa atgcagaaac agattgaaga gatggagaag aagctaaaag aaatccaaac 1380 tactcagcaa gaaagaacag gtgatcagca agaagagacg atgcctacaa aggagacaac 1440 taaactgcaa attgcttccg agtctcagaa aataccagga atgactctat ccagttctgt 1500 ttgtcaagta aactgttgtg ccagagaaac ttcacttgcg gagaacattt ggcaggaaca 1560 acctcattct aaaggtccca gtgtaccttt ctccattttt gatgagtttc ttctttcaga 1620 aaagaagaat aaaagtcctc ctgcagatcc cccacgagtt ttag ctcaac gaagacccct 1680 tgcagttctc aaaacctcag aaagcatcac ctcaaatgaa gatgtgtctc cagatgtttg 1740 tgatgaattt acaggaattg aacccttgag cgaggatgcc attatcacag gcttcagaaa 1800 tgtaacaatt tgtcctaacc cagaagacac ttgtgacttt gccagagcag ctcgttttgt 1860 atccactcct tttcatgaga taatgtcctt gaaggatctc ccttctgatc ctgagagact 1920 gttaccggaa gaagatctag atgtaaagac ctctgaggac cagcagacag cttgtggcac 1980 tatctacagt cagactctca gcatcaagaa gctgagccca attattgaag acagtcgtga 2040 agccacacac tcctctggct tctctggttc ttctgcctcg gttgcaagca cctcctccat 2100 caaatgtctt caaattcctg agaaactaga acttactaat gagacttcag aaaaccctac 2160 tcagtcacca tggtgttcac agtatcgcag acagctactg aagtccctac cagagttaag 2220 tgcctctgca gagttgtgta tagaagacag accaatgcct aagttggaaa ttgagaagga 2280 aattgaatta ggtaatgagg attactgcat taaacgagaa tacctaatat gtgaagatta 2340 caagttattc tgggtggcgc caagaaactc tgcagaatta acagtaataa aggtatcttc 2400 tcaacctgtc ccatgggact tttatatcaa cctcaagtta aaggaacgtt taaatgaaga 2460 ttttgatcat ttttgcagct gttatcaata tcaagatggc tgtattgttt ggcaccaata 2520 tataaactgc ttcacccttc aggatcttct ccaacacagt gaatatatta cccatgaaat 2580 aacagtgttg attatttata accttttgac aatagtggag atgctacaca aagcagaaat 2640 agtccatggt gacttgagtc caaggtgtct gattctcaga aacagaatcc acgatcccta 2700 tgattgtaac aagaacaatc aagctttgaa gatagtggac ttttcctaca gtgttgacct 2760 tagggtgcag ctggatgttt ttaccctcag cggctttcgg actgtacaga tcctggaagg 2820 acaaaagatc ctggctaact gttcttctcc ctaccaggta gacctgtttg gtatagcaga 2880 tttagcacat ttactattgt tcaaggaaca cctacaggtc ttctgggatg ggtccttctg 2940 gaaacttagc caaaatattt ctgagctaaa agatggtgaa ttgtggaata aattctttgt 3000 gcggattctg aatgccaatg atgaggccac agtgtctgtt cttggggagc ttgcagcaga 3060 aatgaatggg gtttttgaca ctacattcca aagtcacctg aacaaagcct tatggaaggt 3120 agggaagtta actagtcctg gggctttgct ctttcagtga 3160 <210> 6 <211> 2673 <212> DNA <213> Artificial Sequence <220> <223> KIF20A cDNA polynucleotide sequence <400> 6 atgtcgcaag ggatcctttc tccgccagcg ggcttgctgt ccgatgacga tgtcgtagtt 60 tctcccatgt ttgagtccac agctgcagat ttggggtctg tggtacgcaa gaacctgcta 120 tcagactgct ctgtcgtctc tacctcccta gaggacaagc agcaggttcc atctgaggac 180 agtatggaga aggtgaaagt atacttgagg gttaggccct tgttaccttc agagttggaa 240 cgacaggaag atcagggttg tgtccgtatt gagaatgtgg agacccttgt tctacaagca 300 cccaaggact cttttgccct gaagagcaat gaacggggaa ttggccaagc cacacacagg 360 ttcacctttt cccagatctt tgggccagaa gtgggacagg catccttctt caacctaact 420 gtgaaggaga tggtaaagga tgtactcaaa gggcagaact ggctcatcta tacatatgga 480 gtcactaact cagggaaaac ccacacgatt caaggtacca tcaaggatgg agggattctc 540 ccccggtccc tggcgctgat cttcaatagc ctccaaggcc aacttcatcc aacacctgat 600 ctgaagccct tgctctccaa tgaggtaatc tggctagaca gcaagcagat ccgacaggag 660 gaaatgaaga agctgtccct gctaaatgga ggcctccaag aggaggagct gtccacttcc 720 ttgaagagga gtgtctacat cgaaagtcgg ataggtacca gcaccagctt cgacagtggc 780 attgctgggc tctcttctat cagtcagtgt accagcagta gccagctgga tgaaacaagt 840 catcgatggg cacagccaga cactgcccca ctacctgtcc cggcaaacat tcgcttctcc 900 atctggatct cattctttga gatctacaac gaactgcttt atgacctatt agaaccgcc t 960 agccaacagc gcaagaggca gactttgcgg ctatgcgagg atcaaaatgg caatccctat 1020 gtgaaagatc tcaactggat tcatgtgcaa gatgctgagg aggcctggaa gctcctaaaa 1080 gtgggtcgta agaaccagag ctttgccagc acccacctca accagaactc cagccgcagt 1140 cacagcatct tctcaatcag gatcctacac cttcaggggg aaggagatat agtccccaag 1200 atcagcgagc tgtcactctg tgatctggct ggctcagagc gctgcaaaga tcagaagagt 1260 ggtgaacggt tgaaggaagc aggaaacatt aacacctctc tacacaccct gggccgctgt 1320 attgctgccc ttcgtcaaaa ccagcagaac cggtcaaagc agaacctggt tcccttccgt 1380 gacagcaagt tgactcgagt gttccaaggt ttcttcacag gccgaggccg ttcctgcatg 1440 attgtcaatg tgaatccctg tgcatctacc tatgatgaaa ctcttcatgt ggccaagttc 1500 tcagccattg ctagccagct tgtgcatgcc ccacctatgc aactgggatt cccatccctg 1560 cactcgttca tcaaggaaca tagtcttcag gtatccccca gcttagagaa aggggctaag 1620 gcagacacag gccttgatga tgatattgaa aatgaagctg acatctccat gtatggcaaa 1680 gaggagctcc tacaagttgt ggaagccatg aagacactgc ttttgaagga acgacaggaa 1740 aagctacagc tggagatgca tctccgagat gaaatttgca atgagatggt agaacagatg 1800 caacagcggg aacagtggtg cagtgaacat ttggacaccc aaaaggaact attggaggaa 1860 atgtatgaag aaaaactaaa tatcctcaag gagtcactga caagttttta ccaagaagag 1920 attcaggagc gggatgaaaa gattgaagag ctagaagctc tcttgcagga agccagacaa 1980 cagtcagtgg cccatcagca atcagggtct gaattggccc tacggcggtc acaaaggttg 2040 gcagct tctg cctccaccca gcagcttcag gaggttaaag ctaaattaca gcagtgcaaa 2100 gcagagctaa actctaccac tgaagagttg cataagtatc agaaaatgtt agaaccacca 2160 ccctcagcca agcccttcac cattgatgtg gacaagaagt tagaagaggg ccagaagaat 2220 ataaggctgt tgcggacaga gcttcagaaa cttggtgagt ctctccaatc agcagagaga 2280 gcttgttgcc acagcactgg ggcaggaaaa cttcgtcaag ccttgaccac ttgtgatgac 2340 atcttaatca aacaggacca gactctggct gaactgcaga acaacatggt gctagtgaaa 2400 ctggaccttc ggaagaaggc agcatgtatt gctgagcagt atcatactgt gttgaaactc 2460 caaggccagg tttctgccaa aaagcgcctt ggtaccaacc aggaaaatca gcaaccaaac 2520 caacaaccac cagggaagaa accattcctt cgaaatttac ttccccgaac accaacctgc 2580 caaagctcaa cagactgcag cccttatgcc cggatcctac gctcacggcg ttccccttta 2640 ctcaaatctg ggccttttgg caaaaagtac taa 2673 <210> 7 <211> 1500 <212> DNA <213> Artificial Sequence <220> <223> CDC20 cDNA polynucleotide sequence <400> 7 atggcacagt tcgcgttcga gagtgacctg cactcgctgc ttcagctgga tgcacccatc 60 cccaatgcac cccctgcgcg ctggcagcgc aaagccaagg aagccgcagg cccggccccc 120 tcacc catgc gggccgccaa ccgatcccac agcgccggca ggactccggg ccgaactcct 180 ggcaaatcca gttccaaggt tcagaccact cctagcaaac ctggcggtga ccgctatatc 240 ccccatcgca gtgctgccca gatggaggtg gccagcttcc tcctgagcaa ggagaaccag 300 cctgaaaaca gccagacgcc caccaagaag gaacatcaga aagcctgggc tttgaacctg 360 aacggttttg atgtagagga agccaagatc cttcggctca gtggaaaacc acaaaatgcg 420 ccagagggtt atcagaacag actgaaagta ctctacagcc aaaaggccac tcctggctcc 480 agccggaaga cctgccgtta cattccttcc ctgccagacc gtatcctgga tgcgcctgaa 540 atccgaaatg actattacct gaaccttgtg gattggagtt ctgggaatgt actggccgtg 600 gcactggaca acagtgtgta cctgtggagt gcaagctctg gtgacatcct gcagcttttg 660 caaatggagc agcctgggga atatatatcc tctgtggcct ggatcaaaga gggcaactac 720 ttggctgtgg gcaccagcag tgctgaggtg cagctatggg atgtgcagca gcagaaacgg 780 cttcgaaata tgaccagtca ctctgcccga gtgggctccc taagctggaa cagctatatc 840 ctgtccagtg gttcacgttc tggccacatc caccaccatg atgttcgggt agcagaacac 900 catgtggcca cactgagtgg ccacagccag gaagtgtgtg ggctgcgctg ggccccagat 960 ggacgacatt tggccagtgg tgg taatgat aacttggtca atgtgtggcc tagtgctcct 1020 ggagagggtg gctgggttcc tctgcagaca ttcacccagc atcaaggggc tgtcaaggcc 1080 gtagcatggt gtccctggca gtccaatgtc ctggcaacag gagggggcac cagtgatcga 1140 cacattcgca tctggaatgt gtgctctggg gcctgtctga gtgccgtgga tgcccattcc 1200 caggtgtgct ccatcctctg gtctccccat tacaaggagc tcatctcagg ccatggcttt 1260 gcacagaacc agctagttat ttggaagtac ccaaccatgg ccaaggtggc tgaactcaaa 1320 ggtcacacat cccgggtcct gagtctgacc atgagcccag atggggccac agtggcatcc 1380 gcagcagcag atgagaccct gaggctatgg cgctgttttg agttggaccc tgcgcggcgg 1440 cgggagcggg agaaggccag tgcagccaaa agcagcctca tccaccaagg catccgctga 1500 1500 <210> 8 <211> 543 <212> DNA <213> Artificial Sequence <220> <223> TK1 cDNA polynucleotide sequence <400> 8 atgagctgca ttaacctgcc cactgtgctg cctggctccc ccagcaagac ccgggggcag 60 atccaggtga ttctcgggcc gatgttctca ggaaaaagca cagagttgat gagacgcgtc 120 cgtcgcttcc agattgctca gtacaagtgc ctggtgatca agtatgccaa agacactcgc 180 tacagcagca gcttctgcac acatgaccgg aacaccatgg aggcactgcc cgcctgcctg 240 ctccgagacg tggcccagga ggccctgggc gtggctgtca taggcatcga cgaggggcag 300 tttttccctg acatcgtgga gttctgcgag gccatggcca acgccgggaa gaccgtaatt 360 gtggctgcac tggatgggac cttccagagg aagccatttg gggccatcct gaacctggtg 420 ccgctggccg agagcgtggt gaagctgacg gcggtgtgca tggagtgctt ccgggaagcc 480 gcctatacca agaggctcgg cacagagaag gagcagattc tgcaatgcag ccctgccaac 540 tga 543 <210> 9 <211> 1395 <212> DNA <213> Artificial Sequence <220> <223> CEP55 cDNA polynucleotide sequence <400> 9 atgtcttcca gaagtaccaa agatttaatt aaaagtaagt ggggatcgaa gcctagtaac 60 tccaaatccg aaactacatt agaaaaatta aagggagaaa ttgcacactt aaagacatca 120 gtggatgaaa tcacaagtgg gaaaggaaag ctgactgata aagagagaca cagacttttg 180 gagaaaattc gagtccttga ggctgagaag gagaagaatg cttatcaact cacagagaag 240 gacaaagaaa tacagcgact gagagaccaa ctgaaggcca gatatagtac taccacattg 300 cttgaacagc tggaagagac aacgagagaa ggagaaagga gggagcaggt gttgaaagcc 360 ttatctgaag agaaagacgt attgaaacaa cagttgtctg ctgcaacctc acgaattgct 420 gaacttgaaa gcaaaaccaa tacactccgt ttatcactccgt acaga ctgtggctcc aaactgcttc 480 aactcatcaa taaataatat tcatgaaatg gaaatacagc tgaaagatgc tctggagaaa 540 aatcagcagt ggctcgtgta tgatcagcag cgggaagtct atgtaaaagg acttttagca 600 aagatctttg agttggaaaa gaaaacggaa acagctgctc attcactccc acagcagaca 660 aaaaagcctg aatcagaagg ttatcttcaa gaagagaagc agaaatgtta caacgatctc 720 ttggcaagtg caaaaaaaga tcttgaggtt gaacgacaaa ccataactca gctgagtttt 780 gaactgagtg aatttcgaag aaaatatgaa gaaacccaaa aagaagttca caatttaaat 840 cagctgttgt attcacaaag aagggcagat gtgcaacatc tggaagatga taggcataaa 900 acagagaaga tacaaaaact cagggaagag aatgatattg ctaggggaaa acttgaagaa 960 gagaagaaga gatccgaaga gctcttatct caggtccagt ttctttacac atctctgcta 1020 aagcagcaag aagaacaaac aagggtagct ctgttggaac aacagatgca ggcatgtact 1080 ttagactttg aaaatgaaaa actcgaccgt caacatgtgc agcatcaatt gcatgtaatt 1140 cttaaggagc tccgaaaagc aagaaatcaa ataacacagt tggaatcctt gaaacagctt 1200 catgagtttg ccatcacaga gccattagtc actttccaag gagagactga aaacagagaa 1260 aaagttgccg cctcaccaaa aagtcccact gctgcactca atgaaagcc t ggtggaatgt 1320 cccaagtgca atatacagta tccagccact gagcatcgcg atctgcttgt ccatgtggaa 1380 tactgttcaa agtag 1395 <210> 10 <211> 2571 <212> DNA <213> Artificial Sequence <220> <223> TTK cDNA polynucleotide sequence <400> 10 atggaatccg aggatttaag tggcagagaa ttgacaattg attccataat gaacaaagtg 60 agagacatta aaaataagtt taaaaatgaa gaccttactg atgaactaag cttgaataaa 120 atttctgctg atactacaga taactcggga actgttaacc aaattatgat gatggcaaac 180 aacccagagg actggttgag tttgttgctc aaactagaga aaaacagtgt tccgctaagt 240 gatgctcttt taaataaatt gattggtcgt tacagtcaag caattgaagc gcttccccca 300 gataaatatg gccaaaatga gagttttgct agaattcaag tgagatttgc tgaattaaaa 360 gctattcaag agccagatga tgcacgtgac tactttcaaa tggccagagc aaactgcaag 420 aaatttgctt ttgttcatat atcttttgca caatttgaac tgtcacaagg taatgtcaaa 480 aaaagtaaac aacttcttca aaaagctgta gaacgtggag cagtaccact agaaatgctg 540 gaaattgccc tgcggaattt aaacctccaa aaaaagcagc tgctttcaga ggaggaaaag 600 aagaatttat cagcatctac ggtattaact gcccaagaat cattttccgg ttcacttggg ga60 cattgg ataggaacaa cagttgtgat tccagaggac agactactaa agccaggttt 720 ttatatggag agaacatgcc accacaagat gcagaaatag gttaccggaa ttcattgaga 780 caaactaaca aaactaaaca gtcatgccca tttggaagag tcccagttaa ccttctaaat 840 agcccagatt gtgatgtgaa gacagatgat tcagttgtac cttgttttat gaaaagacaa 900 acctctagat cagaatgccg agatttggtt gtgcctggat ctaaaccaag tggaaatgat 960 tcctgtgaat taagaaattt aaagtctgtt caaaatagtc atttcaagga acctctggtg 1020 tcagatgaaa agagttctga acttattatt actgattcaa taaccctgaa gaataaaacg 1080 gaatcaagtc ttctagctaa attagaagaa actaaagagt atcaagaacc agaggttcca 1140 gagagtaacc agaaacagtg gcaatctaag agaaagtcag agtgtattaa ccagaatcct 1200 gctgcatctt caaatcactg gcagattccg gagttagccc gaaaagttaa tacagagaaa 1260 cataccactt ttgagcaacc tgtcttttca gtttcaaaac agtcaccacc aatatcaaca 1320 tctaaatggt ttgacccaaa atctatttgt aagacaccaa gcagcaatac cttggatgat 1380 tacatgagct gttttagaac tccagttgta aagaatgact ttccacctgc ttgtcagttg 1440 tcaacacctt atggccaacc tgcctgtttc cagcagcaac agcatcaaat acttgccact 1500 ccacttcaaa atttacaggt tttagcatct tcttcagcaa atgaatgcat ttcggttaaa 1560 ggaagaattt attccatatt aaagcagata ggaagtggag gttcaagcaa ggtatttcag 1620 gtgttaaatg aaaagaaaca gatatatgct ataaaatatg tgaacttaga agaagcagat 1680 aaccaaactc ttgatagtta ccggaacgaa atagcttatt tgaataaact acaacaacac 1740 agtgataaga tcatccgact ttatgattat gaaatcacgg accagtacat ctacatggta 1800 atggagtgtg gaaatattga tcttaatagt tggcttaaaa agaaaaaatc cattgatcca 1860 tgggaacgca agagttactg gaaaaatatg ttagaggcag ttcacacaat ccatcaacat 1920 ggcattgttc acagtgatct taaaccagct aactttctga tagttgatgg aatgctaaag 1980 ctaattgatt ttgggattgc aaaccaaatg caaccagata caacaagtgt tgttaaagat 2040 tctcaggttg gcacagttaa ttatatgcca ccagaagcaa tcaaagatat gtcttcctcc 2100 agagagaatg ggaaatctaa gtcaaagata agccccaaaa gtgatgtttg gtccttagga 2160 tgtattttgt actatatgac ttacgggaaa acaccatttc agcagataat taatcagatt 2220 tctaaattac atgccataat tgatcctaat catgaaattg aatttcccga tattccagag 2280 aaagatcttc aagatgtgtt aaagtgttgt ttaaaaaggg acccaaaaca gaggatatcc 2340 attcctgagc tcctggctca tccat atgtt caaattcaaa ctcatccagt taaccaaatg 2400 gccaagggaa ccactgaaga aatgaaatat gttctgggcc aacttgttgg tctgaattct 2460 cctaactcca ttttgaaagc tgctaaaact ttatatgaac actatagtgg tggtgaaagt 2520 cataattctt catcctccaa gacttttgaa aaaaaaaggg gaaaaaaatg a 2571 <210> 11 <211> 1197 <212> DNA <213> Artificial Sequence <220> <223> CCNB2 cDNA polynucleotide sequence <400> 11 atggcgctgc tccgacgccc gacggtgtcc agtgatttgg agaatattga cacaggagtt 60 aattctaaag ttaagagtca tgtgactatt aggcgaactg ttttagaaga aattggaaat 120 agagttacaa ccagagcagc acaagtagct aagaaagctc agaacaccaa agttccagtt 180 caacccacca aaacaacaaa tgtcaacaaa caactgaaac ctactgcttc tgtcaaacca 240 gtacagatgg aaaagttggc tccaaagggt ccttctccca cacctgagga tgtctccatg 300 aaggaagaga atctctgcca agctttttct gatgccttgc tctgcaaaat cgaggacatt 360 gataacgaag attgggagaa ccctcagctc tgcagtgact acgttaagga tatctatcag 420 tatctcaggc agctggaggt tttgcagtcc ataaacccac atttcttaga tggaagagat 480 ataaatggac gcatgcgtgc catcctagtg gattggctgg tacaagtcca ctccaagttt 540 aggcttctgc aggagactct gtacatgtgc gttggcatta tggatcgatt tttacaggtt 600 cagccagttt cccggaagaa gcttcaatta gttgggatta ctgctctgct cttggcttcc 660 aagtatgagg agatgttttc tccaaatatt gaagactttg tttacatcac agacaatgct 720 tataccagtt cccaaatccg agaaatggaa actctaattt tgaaagaatt gaaatttgag 780 ttgggtcgac ccttgccact acacttctta aggcgagcat caaaagccgg ggaggttgat 840 gttgaacagc acactttagc caagtatttg atggagctga ctctcatcga ctatgatatg 900 gtgcattatc atccttctaa ggtagcagca gctgcttcct gcttgtctca gaaggttcta 960 ggacaaggaa aatggaactt aaagcagcag tattacacag gatacacaga gaatgaagta 1020 ttggaagtca tgcagcacat ggccaagaat gtggtgaaag taaatgaaaa cttaactaaa 1080 ttcatcgcca tcaagaataa gtatgcaagc agcaaactcc tgaagatcag catgatccct 1140 cagctgaact caaaagccgt caaagacctt gcctccccac tgataggaag gtcctag 1197 <210> 12 <211> 2085 <212> DNA <213> Artificial Sequence <220> <223> HJURP cDNA polynucleotide sequence <400> 12 atgctgggta cgctgcgcgc catggagggc gaggacgtgg aagacgacca gctgctgcag 60 aagctcaggg ccagtcgccg ccgcttccag aggcgcatgc agcggctgat agag aagtac 120 aaccagccct tcgaggacac cccggtggtg caaatggcca cgctgaccta cgagacgcca 180 cagggattga gaatttgggg tggaagacta ataaaggaaa gaaacgaagg agagatccag 240 cctgcagtgc ctcaaagccc tttgaaaaat gaattaagaa ggaaatactt gacccaagtg 300 gatatactgc tacaaggtgc agagtatttt gagtgtgcag gtaacagagc tggaagggat 360 gtacgtgtga ctccgctgcc ttcactggcc tcacctgccg tgcctgcccc cggatactgc 420 agtcgtatct ccagaaagag tcctggtgac ccagcgaaac cagcttcatc tcccagagaa 480 tgggatcctt tgcatccttc ctccacagac atggccttag tacctagaaa tgacagcctc 540 tccctacaag agaccagtag cagcagcttc ttaagcagcc agccctttga agatgatgac 600 atttgcaatg tgaccatcag tgacctgtac gcagggatgc tgcactccat gagccggctg 660 ttgagcacaa agccatcaag catcatctcc accaaaacgt tcatcatgca aaactggaac 720 tccaggagga ggcacagata taagagcagg atgaacaaaa catattgcaa aggagccaga 780 cgttctcaga ggagctccaa ggagaacttc ataccctgct ctgagcctgt gaaagggaca 840 ggggcattaa gagattgcaa gaacgtatta gatgtttctt gccgtaagac aggtttaaaa 900 ttggaaaaag cttttcttga agtcaacaga ccccaaatcc ataagttaga tccaagttgg 960 aaggagcgc a aagtgacacc ctcgaagtat tcttccttga tttacttcga ctccagtgca 1020 acatataatc ttgatgagga aaatagattt aggacattaa aatggttaat ttctcctgta 1080 aaaatagttt ccagaccaac aatacgacag ggccatggag agaaccgtca gagggagatt 1140 gaaatccgat ttgatcagct tcatcgggaa tattgcctga gtcccaggaa ccagcctcgc 1200 cggatgtgcc tcccggactc ctgggccatg aacatgtaca gagggggtcc tgcgagtcct 1260 ggtggccttc agggcttaga aacccgcagg ctgagtttac cttccagcaa agcaaaagca 1320 aaaagtttaa gtgaggcttt tgaaaaccta ggcaaaagat ctctggaagc aggtaggtgc 1380 ctgcccaaga gcgattcatc ttcatcactt ccaaagacca accccacaca cagcgcaact 1440 cgcccgcagc agacatctga ccttcacgtt cagggaaata gttctggaat atttagaaag 1500 tcagtgtcac ccagcaaaac tctttcagtc ccagataaag aagtgccagg ccacggaagg 1560 aatcgttacg atgaaattaa agaagaattt gacaagcttc atcaaaagta ttgcctcaaa 1620 tctcctgggc agatgacagt gcctttatgt attggagtgt ctacagataa agcaagtatg 1680 gaagttcgat atcaaacaga aggcttctta ggaaaattaa atccagaccc tcacttccag 1740 ggtttccaga agttgccatc atcacccctg gggtgcagaa aaagtctact gggctcaact 1800 gcaattgagg ctcc ttcatc tacatgtgtt gctcgtgcca tcacgaggga tggcacgagg 1860 gaccatcagt tccctgcaaa aagacccagg ctatcagaac cccagggctc cggacgccag 1920 ggcaattccc tgggtgcctc agatggggtg gacaacaccg tcagaccggg agaccagggc 1980 agctcttcac agcccaactc agaagagaga ggagagaaca cgtcttacag gatggaagag 2040 aaaagtgatt tcatgctaga aaaattggaa actaaaagtg tgtag 2085 <210> 13 <211> 1320 <212> DNA <213> Artificial Sequence <220> <223> NUSAP1 cDNA polynucleotide sequence <400> 13 atgatcatcc cctctctaga ggagctggac tccctcaagt acagtgacct gcagaactta 60 gccaagagtc tgggtctccg ggccaacctg agggcaacca agttgttaaa agccttgaaa 120 ggctacatta aacatgaggc aagaaaagga aatgagaatc aggatgaaag tcaaacttct 180 gcatcctctt gtgatgagac tgagatacag atcagcaacc aggaagaagc tgagagacag 240 ccacttggcc atgtcaccaa aacaaggaga aggtgcaaga ctgtccgtgt ggaccctgac 300 tcacagaatc attcagagat aaaaataagt aatccccactg aattccagaa tcatgaaaag 360 caggaaagcc aggatctcag agctactgca aaagttcctt ctccaccaga cgagcaccaa 420 gaagctgaga atgctgtttc ctcaggtaac agagattcaa aggtaccttc agaaggaaag 480 aaat ctctct acacagatga gtcatccaaa cctggaaaaa ataaaagaac tgcaatcact 540 actccaaact ttaagaagct tcatgaagct cattttaagg aaatggagtc cattgatcaa 600 tatattgaga gaaaaaagaa acattttgaa gaacacaatt ccatgaatga actgaagcag 660 cccatcaata agggaggggt caggactcca gtacctccaa gaggaagact ctctgtggct 720 tctactccca tcagccaacg acgctcgcaa ggccggtctt gtggccctgc aagtcagagt 780 accttgggtc tgaaggggtc actcaagcgc tctgctatct ctgcagctaa aacgggtgtc 840 aggttttcag ctgctactaa agataatgag cataagcgtt cactgaccaa gactccagcc 900 agaaagtctg cacatgtgac cgtgtctggg ggcaccccaa aaggcgaggc tgtgcttggg 960 acacacaaat taaagaccat cacggggaat tctgctgctg ttattacccc attcaagttg 1020 acaactgagg caacgcagac tccagtctcc aataagaaac cagtgtttga tcttaaagca 1080 agtttgtctc gtcccctcaa ctatgaacca cacaaaggaa agctaaaacc atgggggcaa 1140 tctaaagaaa ataattatct aaatcaacat gtcaacagaa ttaacttcta caagaaaact 1200 tacaaacaac cccatctcca gacaaaggaa gagcaacgga agaaacgcga gcaagaacga 1260 aaggagaaga aagcaaaggt tttgggaatg cgaaggggcc tcattttggc tgaagattaa 1320 1320 < 210> 14 < 211 > 4596 <212> DNA <213> Artificial Sequence <220> <223> TOP2A cDNA polynucleotide sequence <400> 14 atggaagtgt caccattgca gcctgtaaat gaaaatatgc aagtcaacaa aataaagaaa 60 aatgaagatg ctaagaaaag actgtctgtt gaaagaatct atcaaaagaa aacacaattg 120 gaacatattt tgctccgccc agacacctac attggttctg tggaattagt gacccagcaa 180 atgtgggttt acgatgaaga tgttggcatt aactataggg aagtcacttt tgttcctggt 240 ttgtacaaaa tctttgatga gattctagtt aatgctgcgg acaacaaaca aagggaccca 300 aaaatgtctt gtattagagt cacaattgat ccggaaaaca atttaattag tatatggaat 360 aatggaaaag gtattcctgt tgttgaacac aaagttgaaa agatgtatgt cccagctctc 420 atatttggac agctcctaac ttctagtaac tatgatgatg atgaaaagaa agtgacaggt 480 ggtcgaaatg gctatggagc caaattgtgt aacatattca gtaccaaatt tactgtggaa 540 acagccagta gagaatacaa gaaaatgttc aaacagacat ggatggataa tatgggaaga 600 gctggtgaga tggaactcaa gcccttcaat ggagaagatt atacatgtat cacctttcag 660 cctgatttgt ctaagtttaa aatgcaaagc ctggacaaag atattgttgc actaatggtc 720 agaagagcat atgatattgc tggatccacc aaagatgtca aagtctttct taatgga aat 780 aaactgccag taaaaggatt tcgtagttat gtggacatgt atttgaagga caagttggat 840 gaaactggta actccttgaa agtaatacat gaacaagtaa accacaggtg ggaagtgtgt 900 ttaactatga gtgaaaaagg ctttcagcaa attagctttg tcaacagcat tgctacatcc 960 aagggtggca gacatgttga ttatgtagct gatcagattg tgactaaact tgttgatgtt 1020 gtgaagaaga agaacaaggg tggtgttgca gtaaaagcac atcaggtgaa aaatcacatg 1080 tggatttttg taaatgcctt aattgaaaac ccaacctttg actctcagac aaaagaaaac 1140 atgactttac aacccaagag ctttggatca acatgccaat tgagtgaaaa atttatcaaa 1200 gctgccattg gctgtggtat tgtagaaagc atactaaact gggtgaagtt taaggcccaa 1260 gtccagttaa acaagaagtg ttcagctgta aaacataata gaatcaaggg aattcccaaa 1320 ctcgatgatg ccaatgatgc agggggccga aactccactg agtgtacgct tatcctgact 1380 gagggagatt cagccaaaac tttggctgtt tcaggccttg gtgtggttgg gagagacaaa 1440 tatggggttt tccctcttag aggaaaaata ctcaatgttc gagaagcttc tcataagcag 1500 atcatggaaa atgctgagat taacaatatc atcaagattg tgggtcttca gtacaagaaa 1560 aactatgaag atgaagattc attgaagacg cttcgttatg ggaagataat gattatgaca 1620 gatcaggacc aagatggttc ccacatcaaa ggcttgctga ttaattttat ccatcacaac 1680 tggccctctc ttctgcgaca tcgttttctg gaggaattta tcactcccat tgtaaaggta 1740 tctaaaaaca agcaagaaat ggcattttac agccttcctg aatttgaaga gtggaagagt 1800 tctactccaa atcataaaaa atggaaagtc aaatattaca aaggtttggg caccagcaca 1860 tcaaag gaag ctaaagaata ctttgcagat atgaaaagac atcgtatcca gttcaaatat 1920 tctggtcctg aagatgatgc tgctatcagc ctggccttta gcaaaaaaca gatagatgat 1980 cgaaaggaat ggttaactaa tttcatggag gatagaagac aacgaaagtt acttgggctt 2040 cctgaggatt acttgtatgg acaaactacc acatatctga catataatga cttcatcaac 2100 aaggaactta tcttgttctc aaattctgat aacgagagat ctatcccttc tatggtggat 2160 ggtttgaaac caggtcagag aaaggttttg tttacttgct tcaaacggaa tgacaagcga 2220 gaagtaaagg ttgcccaatt agctggatca gtggctgaaa tgtcttctta tcatcatggt 2280 gagatgtcac taatgatgac cattatcaat ttggctcaga attttgtggg tagcaataat 2340 ctaaacctct tgcagcccat tggtcagttt ggtaccaggc tacatggtgg caaggattct 2400 gctagtccac gatacatctt tacaatgctc agctctttgg ctcgattgtt atttccacca 2460 aaagatgatc acacgttgaa gtttttatat gatgacaacc agcgtgttga gcctgaatgg 2520 tacattccta ttattcccat ggtgctgata aatggtgctg aaggaatcgg tactgggtgg 2580 tcctgcaaaa tccccaactt tgatgtgcgt gaaattgtaa ataacatcag gcgtttgatg 2640 gatggagaag aacctttgcc aatgcttcca agttacaaga acttcaaggg tactattgaa 2700 gaactggctc c aaatcaata tgtgattagt ggtgaagtag ctattcttaa ttctacaacc 2760 attgaaatct cagagcttcc cgtcagaaca tggacccaga catacaaaga acaagttcta 2820 gaacccatgt tgaatggcac cgagaagaca cctcctctca taacagacta tagggaatac 2880 catacagata ccactgtgaa atttgttgtg aagatgactg aagaaaaact ggcagaggca 2940 gagagagttg gactacacaa agtcttcaaa ctccaaacta gtctcacatg caactctatg 3000 gtgctttttg accacgtagg ctgtttaaag aaatatgaca cggtgttgga tattctaaga 3060 gacttttttg aactcagact taaatattat ggattaagaa aagaatggct cctaggaatg 3120 cttggtgctg aatctgctaa actgaataat caggctcgct ttatcttaga gaaaatagat 3180 ggcaaaataa tcattgaaaa taagcctaag aaagaattaa ttaaagttct gattcagagg 3240 ggatatgatt cggatcctgt gaaggcctgg aaagaagccc agcaaaaggt tccagatgaa 3300 gaagaaaatg aagagagtga caacgaaaag gaaactgaaa agagtgactc cgtaacagat 3360 tctggaccaa ccttcaacta tcttcttgat atgccccttt ggtatttaac caaggaaaag 3420 aaagatgaac tctgcaggct aagaaatgaa aaagaacaag agctggacac attaaaaaga 3480 aagagtccat cagatttgtg gaaagaagac ttggctacat ttattgaaga attggaggct 3540 gttgaagcca aggaaaa aca agatgaacaa gtcggacttc ctgggaaagg ggggaaggcc 3600 aaggggaaaa aaacacaaat ggctgaagtt ttgccttctc cgcgtggtca aagagtcatt 3660 ccacgaataa ccatagaaat gaaagcagag gcagaaaaga aaaataaaaa gaaaattaag 3720 aatgaaaata ctgaaggaag ccctcaagaa gatggtgtgg aactagaagg cctaaaacaa 3780 agattagaaa agaaacagaa aagagaacca ggtacaaaga caaagaaaca aactacattg 3840 gcatttaagc caatcaaaaa aggaaagaag agaaatccct ggtctgattc agaatcagat 3900 aggagcagtg acgaaagtaa ttttgatgtc cctccacgag aaacagagcc acggagagca 3960 gcaacaaaaa caaaattcac aatggatttg gattcagatg aagatttctc agattttgat 4020 gaaaaaactg atgatgaaga ttttgtccca tcagatgcta gtccacctaa gaccaaaact 4080 tccccaaaac ttagtaacaa agaactgaaa ccacagaaaa gtgtcgtgtc agaccttgaa 4140 gctgatgatg ttaagggcag tgtaccactg tcttcaagcc ctcctgctac acatttccca 4200 gatgaaactg aaattacaaa cccagttcct aaaaagaatg tgacagtgaa gaagacagca 4260 gcaaaaagtc agtcttccac ctccactacc ggtgccaaaa aaagggctgc cccaaaagga 4320 actaaaaggg atccagcttt gaattctggt gtctctcaaa agcctgatcc tgccaaaacc 4380 aagaatcgcc gcaaaaggaa gc catccact tctgatgatt ctgactctaa ttttgagaaa 4440 attgtttcga aagcagtcac aagcaagaaa tccaaggggg agagtgatga cttccatatg 4500 gactttgact cagctgtggc tcctcgggca aaatctgtac gggcaaagaa acctataaag 4560 tacctggaag agtcagatga agatgatctg ttttaa 4596 <210> 15 <211> 1578 <212> DNA <213> Artificial Sequence <220> <223> PRC1 cDNA polynucleotide sequence <400> 15 atgaggagaa gtgaggtgct ggcggaggag tccatagtat gtctgcagaa agccctaaat 60 caccttcggg aaatatggga gctaattggg attccagagg accagcggtt acaaagaact 120 gaggtggtaa agaagcatat caaggaagaa ggagagacga ccatcttgca actagaaaaa 180 gatttgcgca cccaagtgga attgatgcga aaacagaaaa aggagagaaa acaggaactg 240 aagctacttc aagagcaaga tcaagaactg tgcgaaattc tttgtatgcc ccactatgat 300 attgacagtg cctcagtgcc cagcttagaa gagctgaacc agttcaggca acatgtgaca 360 actttgaggg aaacaaaggc ttctaggcgt gaggagtttg tcagtataaa gagacagatc 420 atactgtgta tggaagcatt agaccacacc ccagacacaa gctttgaaag agatgtggtg 480 tgtgaagacg aagatgcctt ttgtttgtct ttggagaata ttgcaacact acaaaagttg 540 ctacggcagc tggaaatg tcacaa aatgaagcag tgtgtgaggg gctgcgtact 600 caaatccgag agctctggga caggttgcaa atacctgaag aagaaagaga agctgtggcc 660 accattatgt ctgggtcaaa ggccaaggtc cggaaagcgc tgcaattaga agtggatcgg 720 ttggaagaac tgaaaatgca aaacatgaag aaagtgattg aggcaattcg agtggagctg 780 gttcagtact gggaccagtg cttttatagc caggagcaga gacaagcttt tgcccctttc 840 tgtgctgagg actacacaga aagtctgctc cagctccacg atgctgagat tgtgcggtta 900 aaaaactact atgaagttca caaggaactc tttgaaggtg tccagaagtg ggaagaaacc 960 tggaggcttt tcttagagtt tgagagaaaa gcttcagatc caaatcgatt tacaaaccga 1020 ggaggaaatc ttctaaaaga agaaaaacaa cgagccaagc tccagaaaat gctgcccaag 1080 ctggaagaag agttgaaggc acgaattgaa ttgtgggaac aggaacattc aaaggcattt 1140 atggtgaatg ggcagaaatt catggagtat gtggcagaac aatgggagat gcatcgattg 1200 gagaaagaga gagccaagca ggaaagacaa ctgaagaaca aaaaacagac agagacagag 1260 atgctgtatg gcagcgctcc tcgaacacct agcaagcggc gaggactggc tcccaataca 1320 ccgggcaaag cacgtaagct gaacactacc accatgtcca atgctacggc caatagtagc 1380 attcggccta tctttggagg gacagtctac cactcc cccg tgtctcgact tcctccttct 1440 ggcagcaagc cagtcgctgc ttccacctgt tcagggaaga aaacaccccg tactggcagg 1500 catggagcca acaaggagaa cctggagctc aacggcagca tcctgagtgc gagaactttc 1560 aaaggcttcc aaatctga 1578 <210> 16 <211> 2517 <212> DNA <213> Artificial Sequence <220> <223> TACC3 cDNA polynucleotide sequence <400 > 16 atgagtctgc aggtcttaaa cgacaaaaat gtcagcaatg aaaaaaatac agaaaattgc 60 gacttcctgt tttcgccacc agaagttacc ggaagatcgt ctgttcttcg tgtgtcacag 120 aaagaaaatg tgccacccaa gaacctggcc aaagctatga aggtgacttt tcagacacct 180 ctgcgggatc cacagacgca caggattcta agtcctagca tggccagcaa acttgaggct 240 cctttcactc aggatgacac ccttggactg gaaaactcac acccggtctg gacacagaaa 300 gagaaccaac agctcatcaa ggaagtggat gccaaaacta ctcatggaat tctacagaaa 360 ccagtggagg ctgacaccga cctcctgggg gatgcaagcc cagcctttgg gagtggcagc 540 gccgtggagg aaaaccttag ttcctattcc ttagacagaa gagtgacacc cgcct ctgag 600 accctagaag acccttgcag gacagagtcc cagcacaaag cggagactcc gcacggagcc 660 gaggaagaat gcaaagcgga gactccgcac ggagccgagg aggaatgccg gcacggtggg 720 gtctgtgctc ccgcagcagt ggccacttcg cctcctggtg caatccctaa ggaagcctgc 780 ggaggagcac ccctgcaggg tctgcctggc gaagccctgg gctgccctgc gggtgtgggc 840 acccccgtgc cagcagatgg cactcagacc cttacctgtg cacacacctc tgctcctgag 900 agcacagccc caaccaacca cctggtggct ggcagggcca tgaccctgag tcctcaggaa 960 gaagtggctg caggccaaat ggccagctcc tcgaggagcg gacctgtaaa actagaattt 1020 gatgtatctg atggcgccac cagcaaaagg gcacccccac caaggagact gggagagagg 1080 tccggcctca agcctccctt gaggaaagca gcagtgaggc agcaaaaggc cccgcaggag 1140 gtggaggagg acgacggtag gagcggagca ggagaggacc cccccatgcc agcttctcgg 1200 ggctcttacc acctcgactg ggacaaaatg gatgacccaa acttcatccc gttcggaggt 1260 gacaccaagt ctggttgcag tgaggcccag cccccagaaa gccctgagac caggctgggc 1320 cagccagcgg ctgaacagtt gcatgctggg cctgccacgg aggagccagg tccctgtctg 1380 agccagcagc tgcattcagc ctcagcggag gacacgcctg tggtgcagtt ggcagccgag 1440 ac cccaacag cagagagcaa ggagagagcc ttgaactctg ccagcacctc gcttcccaca 1500 agctgtccag gcagtgagcc agtgcccacc catcagcagg ggcagcctgc cttggagctg 1560 aaagaggaga gcttcagaga ccccgctgag gttctaggca cgggcgcgga ggtggattac 1620 ctggagcagt ttggaacttc ctcgtttaag gagtcggcct tgaggaagca gtccttatac 1680 ctcaagttcg accccctcct gagggacagt cctggtagac cagtgcccgt ggccaccgag 1740 accagcagca tgcacggtgc aaatgagact ccctcaggac gtccgcggga agccaagctt 1800 gtggagttcg atttcttggg agcactggac attcctgtgc caggcccacc cccaggtgtt 1860 cccgcgcctg ggggcccacc cctgtccacc ggacctatag tggacctgct ccagtacagc 1920 cagaaggacc tggatgcagt ggtaaaggcg acacaggagg agaaccggga gctgaggagc 1980 aggtgtgagg agctccacgg gaagaacctg gaactgggga agatcatgga caggttcgaa 2040 gaggttgtgt accaggccat ggaggaagtt cagaagcaga aggaactttc caaagctgaa 2100 atccagaaag ttctaaaaga aaaagaccaa cttaccacag atctgaactc catggagaag 2160 tccttctccg acctcttcaa gcgttttgag aaacagaaag aggtgatcga gggctaccgc 2220 aagaacgaag agtcactgaa gaagtgcgtg gaggattacc tggcaaggat cacccaggag 2280 ggccagag gt accaagccct gaaggcccac gcggaggaga agctgcagct ggcaaacgag 2340 gagatcgccc aggtccggag caaggcccag gcggaagcgt tggccctcca ggccagcctg 2400 aggaaggagc agatgcgcat ccagtcgctg gagaagacag tggagcagaa gactaaagag 2460 aacgaggagc tgaccaggat ctgcgacgac ctcatctcca agatggagaa gatctga 2517 <210> 17 <211> 2715 <212> DNA <213> Artificial Sequence <220> <223 > MCM2 cDNA polynucleotide sequence <400> 17 atggcggaat catcggaatc cttcaccatg gcatccagcc cggcccagcg tcggcgaggc 60 aatgatcctc tcacctccag ccctggccga agctcccggc gtactgatgc cctcacctcc 120 agccctggcc gtgaccttcc accatttgag gatgagtccg aggggctcct aggcacagag 180 gggcccctgg aggaagaaga ggatggagag gagctcattg gagatggcat ggaaagggac 240 taccgcgcca tcccagagct ggacgcctat gaggccgagg gactggctct ggatgatgag 300 gacgtagagg agctgacggc cagtcagagg gaggcagcag agcgggccat gcggcagcgt 360 gaccgggagg ctggccgggg cctgggccgc atgcgccgtg ggctcctgta tgacagcgat 420 gaggaggacg aggagcgccc tgcccgcaag cgccgccagg tggagcgggc cacggaggac 480 ggcgaggagg acgaggagat gatcgagagc atcgagaacc tggaggatct aaggccac 540 tctgtgcgcg agtgggtgag catggcgggc ccccggctgg agatccacca ccgcttcaag 600 aacttcctgc gcactcacgt cgacagccac ggccacaacg tcttcaagga gcgcatcagc 660 gacatgtgca aagagaaccg tgagagcctg gtggtgaact atgaggactt ggcagccagg 720 gagcacgtgc tggcctactt cctgcctgag gcaccggcgg agctgctgca gatctttgat 780 gaggctgccc tggaggtggt actggccatg taccccaagt acgaccgcat caccaaccac 840 atccatgtcc gcatctccca cctgcctctg gtggaggagc tgcgctcgct gaggcagctg 900 catctgaacc agctgatccg caccagtggg gtggtgacca gctgcactgg cgtcctgccc 960 cagctcagca tggtcaagta caactgcaac aagtgcaatt tcgtcctggg tcctttctgc 1020 cagtcccaga accaggaggt gaaaccaggc tcctgtcctg agtgccagtc ggccggcccc 1080 tttgaggtca acatggagga gaccatctat cagaactacc agcgtatccg aatccaggag 1140 agtccaggca aagtggcggc tggccggctg ccccgctcca aggacgccat tctcctcgca 1200 gatctggtgg acagctgcaa gccaggagac gagatagagc tgactggcat ctatcacaac 1260 aactatgatg gctccctcaa cactgccaat ggcttccctg tctttgccac tgtcatccta 1320 gccaaccacg tggccaagaa ggacaacaag gttgctgtag gggaactgac cgatgaagat 1380gtgaagatga tcactagcct ctccaaggat cagcagatcg gagagaagat ctttgccagc 1440 attgctcctt ccatctatgg tcatgaagac atcaagagag gcctggctct ggccctgttc 1500 ggaggggagc ccaaaaaccc aggtggcaag cacaaggtac gtggtgatat caacgtgctc 1560 ttgtgcggag accctggcac agcgaagtcg cagtttctca agtatattga gaaagtgtcc 1620 agccgagcca tcttcaccac tggccagggg gcgtcggctg tgggcctcac ggcgtatgtc 1680 cagcggcacc ctgtcagcag ggagtggacc ttggaggctg gggccctggt tctggctgac 1740 cgaggagtgt gtctcattga tgaatttgac aagatgaatg accaggacag aaccagcatc 1800 catgaggcca tggagcaaca gagcatctcc atctcgaagg ctggcatcgt cacctccctg 1860 caggctcgct gcacggtcat tgctgccgcc aaccccatag gagggcgcta cgacccctcg 1920 ctgactttct ctgagaacgt ggacctcaca gagcccatca tctcacgctt tgacatcctg 1980 tgtgtggtga gggacaccgt ggacccagtc caggacgaga tgctggcccg cttcgtggtg 2040 ggcagccacg tcagacacca ccccagcaac aaggaggagg aggggctggc caatggcagc 2100 gctgctgagc ccgccatgcc caacacgtat ggcgtggagc ccctgcccca ggaggtcctg 2160 aagaagtaca tcatctacgc caaggagagg gtccacccga agctcaacca gatggaccag 2220 gacaag gtgg ccaagatgta cagtgacctg aggaaagaat ctatggcgac aggcagcatc 2280 cccattacgg tgcggcacat cgagtccatg atccgcatgg cggaggccca cgcgcgcatc 2340 catctgcggg actatgtgat cgaagacgac gtcaacatgg ccatccgcgt gatgctggag 2400 agcttcatag acacacagaa gttcagcgtc atgcgcagca tgcgcaagac ttttgcccgc 2460 tacctttcat tccggcgtga caacaatgag ctgttgctct tcatactgaa gcagttagtg 2520 gcagagcagg tgacatatca gcgcaaccgc tttggggccc agcaggacac tattgaggtc 2580 cctgagaagg acttggtgga taaggctcgt cagatcaaca tccacaacct ctctgcattt 2640 tatgacagtg agctcttcag gatgaacaag ttcagccacg acctgaaaag gaaaatgatc 2700 ctgcagcagt tctga 2715 <210> 18 <211> 3627 <212> DNA <213> Artificial Sequence <220> <223> RECQL4 cDNA polynucleotide sequence <400> 18 atggagcggc tgcgggacgt gcgggagcgg ctgcaggcgt gggagcgcgc gttccgacgg 60 cagcgcgggc ggcgaccgag ccaggacgac gtggaggcgg cgccggagga gacccgcgcg 120 ctctaccggg aataccgcac tctgaagcgt accacgggcc aggccggcgg cgggctccgc 180 agctccgagt cgctccccgc ggcggccgaa gaggcgccag agccccgctg ctgggggccc 240 catctgaatc gggctgcgac ca agagtcca cagtctacgc cagggcggag ccgccagggc 300 tcggtgccgg actacgggca gcggctcaag gccaatctga aaggcaccct gcaggccgga 360 ccagccctgg gccgcagacc gtggcctcta ggaagagcct catctaaggc atccacccca 420 aagcccccag gtacagggcc tgtcccctcc tttgcagaaa aagtcagtga tgagcctcca 480 cagctccctg agccccagcc aaggccaggc cggctccagc atctgcaggc atccctgagc 540 cagcggctgg gctccctaga tcctggctgg ttacagcgat gtcacagtga ggtcccagat 600 tttctggggg cccccaaagc ctgcaggcct gatctaggct cagaggaatc acaacttctg 660 atccctggtg agtcggctgt ccttggtcct ggtgctggct cccagggccc agaggcttca 720 gccttccaag aagtcagcat ccgtgtgggg agcccccagc ccagcagcag tggaggcgag 780 aagcggagat ggaacgagga gccctgggag agccccgcac aggtccagca ggagagcagc 840 caagctggac ccccatcgga gggggctggg gctgtagcag ttgaggaaga ccctccaggg 900 gaacctgtac aggcacagcc acctcagccc tgcagcagcc catcgaaccc caggtaccac 960 ggactcagcc cctccagtca agctagggct gggaaggctg agggcacagc ccccctgcac 1020 atcttccctc ggctggcccg ccatgacagg ggcaattacg tacggctcaa catgaagcag 1080 aaacactacg tgcggggccg ggcactccgt agcaggctc c tccgcaagca ggcatggaag 1140 cagaagtggc ggaagaaagg ggagtgtttt gggggtggtg gtgccacagt cacaaccaag 1200 gagtcttgtt tcctgaacga gcagttcgat cactgggcag cccagtgtcc ccggccagca 1260 agtgaggaag acacagatgc tgttgggcct gagccactgg ttccttcacc acaacctgta 1320 cctgaggtgc ccagcctgga ccccaccgtg ctgccactct actccctggg gccctcaggg 1380 cagttggcag agacgccggc tgaggtgttc caggccctgg agcagctggg gcaccaagcc 1440 tttcgccctg ggcaggagcg tgcagtcatg cggatcctgt ctggcatctc cacgctgctg 1500 gtgctgccta caggtgccgg caagtccctg tgctaccagc tcccagcgct gctctacagc 1560 cggcgcagcc cctgcctcac gttggtcgtc tctcccctgc tgtcactcat ggatgaccag 1620 gtgtctggcc tgccaccgtg tctcaaggcg gcctgcatac actcgggcat gaccaggaag 1680 caacgggaat ctgtcctgca gaagattcgg gcagcccagg tacacgtgct gatgctgaca 1740 cctgaggcac tggtgggggc gggaggcctc cctccagccg cacagctgcc tccagttgct 1800 tttgcctgca ttgatgaggc ccactgcctc tcccagtggt cccacaactt ccggccctgc 1860 tacctgcgcg tctgcaaggt gcttcgggag cgcatgggcg tgcactgctt cctgggcctc 1920 acagccacag ccacacgccg cactgccagt gacgtggcac agca cctggc tgtggctgaa 1980 gagcctgacc tccacgggcc agccccagtt cccaccaacc tgcacctttc cgtgtccatg 2040 gacagggaca cagaccaggc actgttgacg ctgctgcaag gcaaacgttt tcaaaacctc 2100 gattccatta tcatttactg caaccggcgc gaggacacag agcggatcgc tgcgctcctc 2160 cgaacctgcc tgcacgcagc ctgggtccca gggtctggag gtcgtgcccc caaaaccaca 2220 gccgaggcct accacgcggg catgtgcagc cgggaacggc ggcgggtaca gcgagccttc 2280 atgcagggcc agttgcgggt ggtggtggcc acggtggcct ttgggatggg gctggaccgg 2340 ccagatgtgc gggctgtgct gcatctgggg ctgcccccaa gcttcgagag ctacgtgcag 2400 gccgtgggcc gggccgggcg tgacgggcag cctgcccact gccacctctt cctgcagccc 2460 cagggcgaag acctgcgaga gctgcgcaga catgtgcacg ccgacagcac ggacttcctg 2520 gctgtgaaga ggctggtaca gcgcgtgttc ccagcctgca cctgcacctg caccaggccg 2580 ccctcggagc aggaaggggc cgtgggtggg gagaggcctg tgcccaagta cccccctcaa 2640 gaggctgagc agcttagcca ccaagcagcc ccaggaccca gaagggtctg catgggccat 2700 gagcgggcac tcccaataca gcttaccgta caggctttgg acatgccgga ggaggccatc 2760 gagactttgc tgtgctacct ggagctgcac ccacaccact ggctggagct gctggcgacc 2820 acctataccc attgccgtct gaactgccct gggggccctg cccagctcca ggccctggcc 2880 cacaggtgtc cccctttggc tgtgtgcttg gcccagcagc tgcctgagga cccagggcaa 2940 ggcagcagct ccgtggagtt tgacatggtc aagctggtgg actccatggg ctgggagctg 3000 gcctctgtgc ggcgggctct ctgccagctg cagtgggacc acgagcccag gacaggtgtg 3060 cggcgtggga caggggtgct tgtggagttc agtgagctgg ccttccacct tcgcagcccg 3120 ggggacttga ccgctgagga gaaggaccag atatgtgact tcctctatgg ccgtgtgcag 3180 gcccgggagc gccaggccct ggcccgtctg cgcagaacct tccaggcctt tcacagcgta 3240 gccttcccca gctgcgggcc ctgcctggag cagcaggatg aggagcgcag caccaggctc 3300 aaggacctgc tcggccgcta ctttgaggaa gaggaagggc aggagccggg aggcatggag 3360 gacgcacagg gccccgagcc agggcaggcc agactccagg attgggagga ccaggtccgc 3420 tgcgacatcc gccagttcct gtccctgagg ccagaggaga agttctccag cagggctgtg 3480 gcccgcatct tccacggcat cggaagcccc tgctacccgg cccaggtgta cgggcaggac 3540 cgacgcttct ggagaaaata cctgcacctg agcttccatg ccctggtggg cctggccacg 3600 gaagagctcc tgcaggtggc ccgctga 3627 <210> 19 <211> 3582 <212> DNA <213> Artificial Sequence <220> <223> SPAG5 cDNA polynucleotide sequence <400> 19 atgtggcgag tgaaaaaact gagcctcagc ctgtcgcctt cgccccagac gggaaaacca 60 tctatgagaa ctcctctccg tgaacttacc ctgcagcccg gtgccctcac caactctgga 120 aaaagatccc ccgcttgctc ctcgctgacc ccatcactgt gcaagctggg gctgcaggaa 180 ggcag caaca actcatctcc agtggatttt gtaaataaca agaggacaga cttatcttca 240 gaacatttca gtcattcctc aaagtggcta gaaacttgtc agcatgaatc agatgagcag 300 cctctagatc caattcccca aattagctct actcctaaaa cgtctgagga agcagtagac 360 ccactgggca attatatggt taaaaccatc gtccttgtac catctccact ggggcagcaa 420 caagacatga tatttgaggc ccgtttagat accatggcag agacaaacag catatcttta 480 aatggacctt tgagaacaga cgatctggtg agagaggagg tggcaccctg catgggagac 540 aggttttcag aagttgctgc tgtatctgag aaacctatct ttcaggaatc tccgtcccat 600 ctcttagagg agtctccacc aaatccctgt tctgaacaac tacattgctc caaggaaagc 660 ctgagcagta gaactgaggc tgtgcgtgag gacttagtac cttctgaaag taacgccttc 720 ttgccttcct ctgttctctg gctttcccct tcaactgcct tggcagcaga tttccgtgtc 780 aatcatgtgg acccagagga ggaaattgta gagcatggag ctatggagga aagagaaatg 840 aggtttccca cacatcctaa ggagtctgaa acagaagatc aagcacttgt ctcaagtgtg 900 gaagatattc tgtccacatg cctgacacca aatctagtag aaatggaatc ccaagaagct 960 ccaggcccag cagtagaaga tgttggtagg attcttggct ctgatacaga gtcttggatg 1020 tccccactgg cctggctgga aa aaggtgta aatacctccg tcatgctgga aaatctccgc 1080 caaagcttat cccttccctc gatgcttcgg gatgctgcaa ttggcactac ccctttctct 1140 acttgctcgg tggggacttg gtttactcct tcagcaccac aggaaaagag tacaaacaca 1200 tcccagacag gcctggttgg caccaagcac agtacttctg agacagagca gctcctgtgt 1260 ggccggcctc cagatctgac tgccttgtct cgacatgact tggaagataa cctgctgagc 1320 tctcttgtca ttctggaggt tctctcccgc cagcttcggg actggaagag ccagctggct 1380 gtccctcacc cagaaaccca ggacagtagc acacagactg acacatctca cagtgggata 1440 actaataaac ttcagcatct taaggagagc catgagatgg gacaggccct acagcaggcc 1500 agaaatgtca tgcaatcatg ggtgcttatc tctaaagagc tgatatcctt gcttcaccta 1560 tccctgttgc atttagaaga agataagact actgtgagtc aggagtctcg gcgtgcagaa 1620 acattggtct gttgctgttt tgatttgctg aagaaattga gggcaaagct ccagagcctc 1680 aaagcagaaa gggaggaggc aaggcacaga gaggaaatgg ctctcagagg caaggatgcg 1740 gcagagatag tgttggaggc tttctgtgca cacgccagcc agcgcatcag ccagctggaa 1800 caggacctag catccatgcg ggaattcaga ggccttctga aggatgccca gacccaactg 1860 gtagggcttc atgccaagca agaagagc tg gttcagcaga cagtgagtct tacttctacc 1920 ttgcaacaag actggaggtc catgcaactg gattatacaa catggacagc tttgctgagt 1980 cggtcccgac aactcacaga gaaactcaca gtcaagagcc agcaagccct gcaggaacgt 2040 gatgtggcaa ttgaggaaaa gcaggaggtt tctagggtgc tggaacaagt ctctgcccag 2100 ttagaggagt gcaaaggcca aacagaacaa ctggagttgg aaaacagtcg tctagcaaca 2160 gatctccggg ctcagttgca gattctggcc aacatggaca gccagctaaa agagctacag 2220 agtcagcata cccattgtgc ccaggacctg gctatgaagg atgagttact ctgccagctt 2280 acccagagca atgaggagca ggctgctcaa tggcaaaagg aagagatggc actaaaacac 2340 atgcaggcag aactgcagca gcaacaagct gtcctggcca aagaggtgcg ggacctgaaa 2400 gagaccttgg agtttgcaga ccaggagaat caggttgctc acctggagct gggtcaggtt 2460 gagtgtcaat tgaaaaccac actggaagtg ctccgggagc gcagcttgca gtgtgagaac 2520 ctcaaggaca ctgtagagaa cctaacggct aaactggcca gcaccatagc agataaccag 2580 gagcaagatc tggagaaaac acggcagtac tctcaaaagc tagggctgct gactgagcaa 2640 ctacagagcc tgactctctt tctacagaca aaactaaagg agaagactga acaagagacc 2700 cttctgctga gtacagcctg tcctcccacc cag gaacacc ctctgcctaa tgacaggacc 2760 ttcctgggaa gcatcttgac agcagtggca gatgaagagc cagaatcaac tcctgtgccc 2820 ttgcttggaa gtgacaagag tgctttcacc cgagtagcat caatggtttc ccttcagccc 2880 gcagagaccc caggcatgga ggagagcctg gcagaaatga gtattatgac tactgagctt 2940 cagagtcttt gttccctgct acaagagtct aaagaagaag ccatcaggac tctgcagcga 3000 aaaatttgtg agctgcaagc taggctgcag gcccaggaag aacagcatca ggaagtccag 3060 aaggcaaaag aagcagacat agagaagctg aaccaggcct tgtgcttgcg ctacaagaat 3120 gaaaaggagc tccaggaagt gatacagcag cagaatgaga agatcctaga acagatagac 3180 aagagtggcg agctcataag ccttagagag gaggtgaccc accttacccg ctcacttcgg 3240 cgtgcggaga cagagaccaa agtgctccag gaggccctgg caggccagct ggactccaac 3300 tgccagccta tggccaccaa ttggatccag gagaaagtgt ggctctctca ggaggtggac 3360 aaactgagag tgatgttcct ggagatgaaa aatgagaagg aaaaactcat gatcaagttc 3420 cagagccata gaaatatcct agaggagaac cttcggcgct ctgacaagga gttagaaaaa 3480 ctagatgaca ttgttcagca tatttataag accctgctct ctattccaga ggtggtgagg 3540ggatgcaaag aactacaggg attgctggaa tttctgagct aa 3582
Claims (10)
UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 및 SPAG5로 이루어진 군으로부터 선택되는 하나 이상의 유전자를 더 포함하는 것을 특징으로 하는 비근침윤성 방광암의 예후 예측용 바이오마커 조성물.According to claim 1,
Characterized in that it further comprises one or more genes selected from the group consisting of UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 and SPAG5 A biomarker composition for predicting the prognosis of non-muscle invasive bladder cancer.
UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 및 SPAG5로 이루어진 군으로부터 선택되는 하나 이상의 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 측정하는 제제를 추가로 포함하는 것을 특징으로 하는 비근침윤성 방광암의 예후 예측용 조성물.According to claim 3,
mRNA of one or more genes selected from the group consisting of UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 and SPAG5, or to the gene A composition for predicting the prognosis of non-muscle invasive bladder cancer, characterized in that it further comprises an agent for measuring the expression level of the protein encoded by.
mRNA의 발현수준을 측정하는 제제는 선택된 유전자에 특이적으로 결합하는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브이며; 단백질의 발현수준을 측정하는 제제는 선택된 유전자로부터 코딩되는 단백질에 특이적인 항체인 것을 특징으로 하는 비근침윤성 방광암의 예후 예측용 조성물.According to claim 3,
Agents for measuring mRNA expression levels are antisense oligonucleotides, primer pairs or probes that specifically bind to a selected gene; A composition for predicting the prognosis of non-muscle invasive bladder cancer, characterized in that the agent for measuring the expression level of the protein is an antibody specific to the protein encoded by the selected gene.
상기 단계는 UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, RECQL4 및 SPAG5로 이루어진 군으로부터 선택되는 하나 이상의 유전자의 mRNA 또는 상기 유전자에 의해 코딩되는 단백질의 발현수준을 더 측정하는 것을 특징으로 하는 비근침윤성 방광암의 예후 예측을 위한 정보를 제공하는 방법.According to claim 8,
The step is UHRF1, AURKB, TPX2, BUB1B, KIF20A, CDC20, TK1, CEP55, TTK, CCNB2, HJURP, NUSAP1, TOP2A, PRC1, TACC3, MCM2, mRNA of one or more genes selected from the group consisting of RECQL4 and SPAG5, or A method for providing information for predicting the prognosis of non-muscle invasive bladder cancer, characterized in that further measuring the expression level of the protein encoded by the gene.
상기 생물학적 시료는 환자의 조직, 세포, 혈액, 혈청, 혈장, 타액 및 소변으로 이루어진 군에서 선택되는 것을 특징으로 하는 비근침윤성 방광암의 예후 예측을 위한 정보를 제공하는 방법.According to claim 8,
The biological sample is a method for providing information for predicting the prognosis of non-muscle invasive bladder cancer, characterized in that selected from the group consisting of tissue, cells, blood, serum, plasma, saliva and urine of the patient.
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| KR20160082028A (en) | 2014-12-30 | 2016-07-08 | 충북대학교 산학협력단 | Use of RSPH9 for Prognostic Marker Diagnosis of Bladder Cancer |
| KR20190089552A (en) | 2018-01-23 | 2019-07-31 | 충북대학교 산학협력단 | Biomarkers for diagnosis of Non-muscle invasive bladder cancer and uses thereof |
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| KR20160082028A (en) | 2014-12-30 | 2016-07-08 | 충북대학교 산학협력단 | Use of RSPH9 for Prognostic Marker Diagnosis of Bladder Cancer |
| KR20190089552A (en) | 2018-01-23 | 2019-07-31 | 충북대학교 산학협력단 | Biomarkers for diagnosis of Non-muscle invasive bladder cancer and uses thereof |
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