、】】Litlekalsoy et al. BMC Cancer (2016) 16:549 DOI 10.1186/s12885-016-2580-y
RESEARCH ART ICLE
Open Access Expression of circadian clock genes and proteinsinurothelialcancerisrelatedto cancer-associated genes 尿道上皮癌中昼夜节律基因和蛋白质的表达与癌症相关基因有关
Jorunn Litlekalsoy1,2,7* , Kari Rostad3, Karl-Henning Kalland1,4, Jens G. Hostmark2,5 and Ole Didrik Laerum1,6
Abstract Background: The purpose of this study was to evaluate invasive and metastatic potential of urothelial cancer by investigating differential expression of various clock genes/proteins participating in the 24 h circadian rhythms and to compare these gene expressions with transcription of other cancer-associated genes. 这项研究的目的是通过调查参与24 h昼夜节律的各种钟基因/蛋白质的表达差异,并比较这些基因表达与其他癌症相关基因的转录，从而评估尿路上皮癌的侵袭性和转移潜力。 Methods:Twenty seven paired samples of tumour and benign tissue collected from patients who underwent cystectomy were analysed and compared to 15 samples of normal bladder tissue taken from patients who underwent cystoscopy for benign prostate hyperplasia (unrelated donors). Immunohistochemical analyses were made for clock and clock-related proteins. In addition, the gene-expression levels of 22 genes (clock genes, casein kinases,oncogenes,tumoursuppressorgenesandcytokeratins)wereanalysedbyreal-timequantitativePCR(qPCR). 从经历膀胱切除术的患者身上收集的肿瘤和良性组织的二十七个配对样品进行分析，并与取自经历过用于良性前列腺增生的膀胱镜检查的患者（不相关供体）的正常膀胱组织的15个样品进行比较。对时钟和时钟相关蛋白进行免疫组织化学分析。另外，通过实时定量PCR（qPCR）分析22种基因（钟基因，酪蛋白激酶，致癌基因，肿瘤抑制基因和细胞角蛋白）的基因表达水平. Results:Considerable up- or down-regulation and altered cellular distribution of different clock proteins, a reductionofcaseinkinase1A1(CSNK1A1)andincreaseofcaseinkinasealpha1E(CSNK1E)werefound.Thepattern was significantly correlated with simultaneous up-regulation of stimulatory tumour markers, and a down-regulation of several suppressor genes. The pattern was mainly seen in aneuploid high-grade cancers. Considerablealterations were also found in the neighbouring bladder mucosa. 发现不同时钟蛋白的大量上调或下调和改变的细胞分布，酪蛋白激酶1A1（CSNK1A1）的减少和酪蛋白激酶α1E（CSNK1E）的增加。该模式与刺激性肿瘤标志物的同时上调和几种抑制基因的下调显着相关。该模式主要见于非整倍体高级癌症。在相邻的膀胱粘膜中也发现了相当大的改变。 Conclusions:The close correlation between altered expression of various clock genes and common tumour markers in urothelial cancer indicates that disturbed function in the cellular clock work may be an important additional mechanism contributing to cancer progression and malignant behaviour. 尿路上皮癌中各种时钟基因和常见肿瘤标志物的表达的改变之间的密切相关性表明在细胞时钟工作中的干扰功能可能是促进癌症进展和恶性表现的重要的另外机制。 Keywords: Circadian clock genes, Casein kinases, Oncogenes, Tumour suppressor genes and cytokeratins 生物钟基因，酪蛋白激酶，癌基因，肿瘤抑制基因和细胞角蛋白
Background
Time is a fundamental part of all biological processes in tissues and cells. Both in rodents and humans, the circadian timing system affects many cellular and physiological functions, including cell proliferation, metabolic pathways, protein synthesis and energy me- tabolism [1]. Severe and prolonged disturbances of the circadian timing system are believed to predispose to cancer development in different organs, not only in the mammary and prostate glands, but also in several other types of cancer, including ovarian, kidney, brain, colorectal,
* Correspondence: Jorunn.Litlekalsoy@k1.uib.no 1Department of Clinical Science, The Gade Laboratory of Pathology, University of Bergen, Bergen, Norway 2Department of Clinical Medicine, Section of Surgery, University of Bergen, Bergen, Norway
Full list of author information is available at the end of the article
lung, head/neck, pancreatic cancer and hematological malignancies [2–14]. 时间是组织和细胞中所有生物进程的基本部分。在啮齿动物和人类，昼夜节律系统影响许多细胞和生理功能，包括细胞增殖，代谢途径，蛋白质合成和能量代谢[1]。认为昼夜节律计时系统的严重和持久的紊乱易于导致不同器官中的癌症发展，不仅在乳腺和前列腺中，而且在几种其他类型的癌症中，包括卵巢，肾，脑，结肠直肠，肺，头 /颈，胰腺癌和血液恶性肿瘤[2-14]。
The mammalian circadian clock system consists of positive and negative regulators, with a complex auto- regulatory transcriptional and translational feedback program. By accumulating and binding to the promoter region of the two transcriptions factors, BMAL1 and CLOCK, PER and CRY proteins reduce the transcription of many genes, including their own. This occurs during ambient light exposure via the master clock in the brain, the suprachiasmaticus nucleus (SCN). The correspond- ing proteins oscillate with a delayed phasing and with maximum levels at dusk [15]. 哺乳动物昼夜节律钟系统由正和负调节器组成，具有复杂的自调节转录和翻译反馈程序。通过积累和绑定到两个转录因子的启动子区域，BMAL1和CLOCK，PER和CRY蛋白减少许多基因的转录，包括它们自己。这在环境光暴露期间通过大脑中的主时钟，视交叉上核（SCN）发生。相应的蛋白质以延迟相位振荡，在黄昏时具有最大水平[15]。
The transcription factors CLOCK and BMAL1 form a heterodimerwhichinhumansisactingstimulatoryongene transcription during night time. CLOCK also contributes
? 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Litlekalsoyetal.BMCCancer(2016)16:549
to chromatin-remodelling and mediates acetylation of BMAL1. The type of phasing can vary from organ to organ. For instance, BMAL1 undergoes rhythmic acetylationin the liver where the timing parallels the down-regulation of circadian transcription in clock-controlled genes. 转录因子CLOCK和BMAL1形成异源二聚体，对人在
夜间的基因转录起刺激作用。 CLOCK还有助于染色质重塑和调解BMAL1的乙酰化。相位的类型可以随器官而变化。例如，BMAL1在肝脏中经历节律性乙酰化，其中时间与钟基因控制下昼夜节律转录的下调相应。 The 24 h clock generation is modified by post- translational events such as phosphorylation and ubiquiti- nation which contribute to precision, stability and nuclear translocation of the core clock proteins. PER and BMAL1 have also been identified as tumour suppressors [15–20]. Casein kinase 1 epsilon and delta (CSNK1E and CSNK1D) are critical in regulating the core circadian protein turnover inmammals. Mutations in either of these kinases may thus have dramatic effects on the circadian period [21]. 24 h时钟的生成由翻译后事件修改，如磷酸化和泛素
化，这有助于核心时钟蛋白质的精度，稳定性和核易位。PER和BMAL1也被鉴定为肿瘤抑制剂[15-20]。酪蛋白
激酶1ε和δ（CSNK1E和CSNK1D）是调节哺乳动物昼夜节律核心蛋白周转的关键。因此这些激酶中的任一种
的突变都可以对昼夜周期具有显着的影响力[21]。
Urothelial carcinoma of the bladder is a very complex malignancy with multiple alterations in complementary pathways. The advent of high-throughput methods of molecular analysis, as microarray-based approaches, has been used extensively to look for expression profiles in effort to sub-classify bladder cancer (stage and pathways) and to predict outcomes and response to systemic treat- ments. Several tissue and blood-based biomarkers have been identified, but status as of today is that no biomarker panel is yet validated for individual prognostic and daily clinical practice. A problem is that most researchers com- bine biomarkers from a single pathway (cell-cycle, apop- tosis or angiogenesis) while the focus rather should be in investigating biomarker combinations that encompass a variety of different pathways to increase the predictive value and opportunity for targeted treatment. Standard pathological features and imaging are insufficient to allow accurate staging, prognostication and prediction of the pa- tient’s outcome [22, 23]. This reveals an urgent need for identifying novel biomarkers that can define the invasive urothelial carcinomas with intrinsic property for recur- rence and metastases. 膀胱的尿路上皮癌是一种非常复杂的恶性肿瘤，在互补途径中具有多种改变。分子分析的高通量方法作为基于微阵列的方法的出现已广泛用于寻找表达谱，以努力亚细分膀胱癌（阶段和途径）并预测结果和对全身治疗的反应。已经鉴定了几种基于组织和血液的生物标志物，但是至今的状态是，尚未验证生物标志物组用于个体预后和日常临床实践。一个问题是大多数研究人员结合来自单一途径（细胞周期，凋亡或血管生成）的生物标志物，而焦点应当在于研究包含多种不同途径的生物标志
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物组合，以增加靶向治疗的预测价值和机会。标准病理特
征和成像不足以允许准确的分期，预后和预测患者的结果[22,23]。这揭示了迫切需要鉴定新的生物标志物，可以定义浸润性尿路上皮癌与复发和转移的固有属性。
The urinary system undergoes significant circadian rhythms in humans. During day and night both urinary excretion and extrusion of urine are actively regulated by several internal factors, such as antidiuretic hormone [24]. Such circadian variations led us to postulate that similar toother organs, perturbation of the clockwork may be a contributory mechanism of dysregulation during the devel- opment of urothelial cancer. Since clock genes have a modifying role in the gene regulation, they mayinteract with the transcription of oncogenes and/or tumour suppressor-genes. If so, they might be used as independent or additional markers of malignant behaviour. Therefore, ten key proteins of the clockwork were selected for a com- bined analysis of transcriptional activity and presence of their proteins in the malignant cells. For comparison, simultaneous analyses of gene-expression patterns were performed for oncogenes and suppressor-genes that are commonly altered in urothelial cancer. 泌尿系统在人类中经历显着的昼夜节律。在白天和晚上，
尿排泄和尿排出都受到几种内在因素的积极调节，如抗利尿激素[24]。这样的昼夜变化导致我们假设类似于其他器
官，钟表机构的扰动可能是泌尿上皮癌发展期间的失调的一个促成机制。由于时钟基因在基因调节中具有修饰作用，它们可以与癌基因和/或肿瘤抑制基因的转录相互作用。如果是这样，它们可以用作恶性行为的独立或另外的标志物。因此，选择钟表机构的十个关键蛋白质用于组合分析转录活性和它们的蛋白质在恶性细胞中的存在。为了比较，
对通常在尿路上皮癌中改变的致癌基因和抑制基因进行基
因表达模式的同时分析。
Methods Patient material and tissue患者材料和组织
Twenty-seven patients with invasive urothelial cancer undergoing cystectomy from 2006 to 2009 were included. General procedures for the cystectomy patients are thatthe patients enter the operating room around 07:45 in the morning. The anesthesia is completed around 08:20 and within the next 5–10 min open surgery is performed. The bladder is removed from the body around 10:00 where- upon the surgeon immediately collects tissue samples from tumour and adjacent normal appearing mucosa into separate tubes. Within twenty minutes, the harvested bladder biopsies are cut into small pieces and snap frozen at ?80 °C. Patient details are given in Table 1. Normal bladder biopsies were taken from 15 male patients who had TUR-P (transurethral resection of the prostate) for benign prostatic hyperplasia (BPH). The mucosal biopsies consisted of the whole urothelial layer and some under- lying connective tissue. A major part of the cell nuclei were from urothelium as compared to sub-mucosal fibro- blasts. Both the cystectomies and the unrelated normal mucosa were harvested in the time period 9 to 12 AM. Paraffin-embeddedtissueslidesweremadeforhistological diagnostics, and classified by the WHO and NM-system. The study was approved by the Regional Ethical Commit- tee(REKNo.12226/REKNo.2009/1527). Litlekalsoyetal.BMCCancer(2016)16:549
包括2006年至2009年进行膀胱切除术的侵入性尿路上皮癌的二十七名患者。膀胱切除术患者的一般程序是患者在早上大约07:45进入手术室。麻醉在大约08:20完成，并且在接下来的5-10分钟内进行开放手术。膀胱在约10:00时从身体移除，其中外科医生立即从肿瘤和相邻正常出现的粘膜收集组织样品到单独的管中。在20分钟内，将收获的膀胱活检切成小块，并在-80℃快速冷冻。患者细节在表1中给出。正常膀胱活检取自具有TUR-P（前列腺的经尿道切除术）的15位男性患者用于良性前列腺增生（BPH）。粘膜活检由整个尿路上皮层和一些下层结缔组织组成。与粘膜下成纤维细胞相比，细胞核的主要部分来自尿道上皮。在9至12AM的时间段收获两个囊肿切除术和不相关的正常粘膜。制备石蜡包埋的组织切片用于组织学诊断，并通过WHO和NM-系统分类。该研究由区域伦理委员会批准（REK第12226 / REK第2009/1527号）。
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Immunohistochemistry免疫组织化学
The paraffin blocks were cut in 5 μm sections andstained with antibodies listed in Table 2. The sections were de-paraffinised and pre-treated as listed in Table 2, and stained as described earlier [25]. Sections of tissue microarrays made of twelve different tissues, reported to express one or more of our chosen proteins, served as control. 将石蜡块切成5μm的切片并用表2中列出的抗体染色。如表2所列，将切片脱石蜡并进行预处理，并如前所述进行染色[25]。由报道表达一种或多种我们选择的蛋白质的十二种不同组织制成的组织微阵列切片用作对照。
Evaluation of staining results
The analyses were made separately for the tumour and neighbouring benign tissue from cystectomies, and unre- lated normal mucosa. Positive staining of epithelial cells was estimated as weakly, moderately and strong, (separ- ately for the nucleus (N) and the cytoplasm (C)). Count- ing was performed on cells from tumour, normal appearing mucosa without atypia, and normal mucosa from the 15 individuals (Table 3). For control, the same staining procedure was performed on tissue microarrays comprising other human tumours/normal tissues. Allcases were scored on coded specimens separately byODL andJGH. 分别对来自囊肿切除术的肿瘤和邻近良性组织和未释放的正常粘膜进行分析。上皮细胞的阳性染色估计为弱，中等和强（细胞核（N）和细胞质（C）分开）。对来自肿瘤，没有异型的正常出现的粘膜和来自15个个体的正常粘膜的细胞进行计数（表3）。对于对照，对包含其他人肿瘤/正常组织的组织微阵列进行相同的染色程序。通过ODL和JGH分别对编码的标本进行所有病例的评分。
Flow cytometry (FCM)流式细胞术（FCM）
FCM was performed on single cell suspensions of tumour tissue obtained by cutting the tissue into small pieces which were shaken, filtered, spun down, re-suspended in
Litlekalsoyetal.BMCCancer(2016)16:549 Page 4of 17
Table 1 Tumour grade, invasiveness, T-stage, ploidy and survival in the individual patients 表1各个患者的肿瘤等级，侵袭性，T阶段，倍性和存活率
No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
G Low High High Low High Low High Low High High Low High Low High High High High Low High High High High Low High Low Low High
V.I.
pTa
pT1
pT2A x
x
pT2B
pT3B
D x
A
D-S 4,22
x x
x x x
7,25 - 7,7 8 3,5
x
x
12,89 -
x x
x
72 7,91 7,2
x x x x x x x x x x x
x
x
8,13 6,86 8,2 6,2 12 14 4,4 64 24 18 84 18
x x
x
41 41 3,3
x
13
A-S
Survival A/D A
1,9 17
D/1m D/10m A A A
23,04 - 10,06 21
D/6m A A D/10m A
26,2 - 32 28 21 20 - 15 18 18 20 17 38 15
D/14m A A A A A D/11m A D/17m A D/3m A D/10m A D/24m
20
D/10m
x
x
x
x
x x
x
x
x
x
x
x
x
x
x x x x x x x
x
x
x
x
x
x x
x x x
x
x
x x
x
No case number, G grade, V.I. vascular invasion, pTa-pT1-pT2A-pT2B-pT3B tumour stage, D Diploid, A Aneuploid, D-S Diploid S-phase, A-S Aneuploid S-phase, Survival A/D survival after surgery (in months, m), A alive, D dead
无病例编号，G级，V.I。血管侵袭，pTa-pT1-pT2A-pT2B-pT3B肿瘤期，D二倍体，A非整倍体，D-S二倍体S期，A-S非整倍体S期，
生存A / D生存手术后（月，m），A活，D死
RNA提取和实时定量PCR（qPCR） RNA纯化和单链cDNA合成 Biopsies were ground to powder under liquid N2. Total RNA was extracted according to standard protocols (Invitrogen Trizol LS protocol and Qiagen miRNeasy protocol; Invitrogen, Carson City, CA). 30 μl of single- stranded cDNA for qPCR analysis was synthesised from 1 μg of total RNA according 对通过将组织切成小片获得的肿瘤组织的单细胞悬液进to Ambion (Ambion, TX, USA) instructions. PBS and fixed by addition of 96 % ethanol, stained with propidium iodide as earlier described [26] and analysed on a FACScan flow cytometer (Becton Dickinson, Palo Alto, CA, USA). Normal human lymphocytes were used as standard, and the ploidy index (PI) was calculated as a ratio between the peak channel for the tumour cells and the peak channel for thelymphocytes.
行FCM，将其振摇，过滤，离心，重悬于PBS中并通过加入96％乙醇固定，如前所述用碘化丙啶染色[26 并在FACScan流式细胞仪（Becton Dickinson，Palo Alto，CA，USA）上分析。使用正常人淋巴细胞作为标准，计算作为肿瘤细胞的峰值通道与淋巴细胞的峰值通道之间的比率的倍性指数（PI）。
RNA extraction and real-time quantitative PCR (qPCR) RNA purification and single-stranded cDNA synthesis 将活检样品在液氮下研磨成粉末。根据标准方案（Invitrogen Trizol LS方案和Qiagen miRNeasy方案; Invitrogen，Carson City，CA）提取总RNA。根据Ambion（Ambion，TX，USA）说明，从1μg总RNA合成30μl用于qPCR分析的单链cDNA。
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