Expression of circadian clock genes and(5)

表8时钟基因和常见肿瘤标记物之间的相关性

Table 8 Correlations between the clock genes and common tumour markers

Genes encoding BMAL

-TP53 - P16 - PTEN - KRAS - NRAS

p-value 0.031 0.001 0.029 0.000 0.000

Correlation coeff, C 0.415 0.601 0.419 0.670 0.694

Genes encoding CSNK1A1

-TP53 -PTEN -KRAS -NRAS -UPAR -PAI-1 -KRT7 -KRT5 - P16 -EGFR -NRAS -TP53 - P16 -PTEN -KRAS -NRAS -UPAR -PAI-1 -KRT7

p-value 0.005 0.001 0.004 0.000 0.001 0.003 0.007 0.005 0.040 0.035 0.001 0.008 0.013 0.021 0.008 0.000 0.010 0.033 0.033

Litlekalsoyetal.BMCCancer(2016)16:549 Page 16of 17

Correlation coeff, C 0.527 0.582 0.536 0.635 0.611 0.546 0.504 0.521 0.397 0.407 0.584 0.499 0.471 0.442 0.497 0.659 0.487 0.412 0.412

CLOCK PER1 -KRAS - NRAS -UPAR - PAI-1

0.000 0.005 0.031 0.047

0.634 0.525 0.416 0.386

CRY-1

PER2 -PTEN - PAI-1 - KRT7 - KRT5 - KRT10

0.017 0.011 0.011 0.044 0.034

0.455 0.480 0.480 0.390 0.409

CSNK1E

PER3 -KRT5 - KRT14

0.019 0.045

0.448 0.389

Thecorrelationcoefficients:C<0.3:poorcorrelation,0.3

相关系数：C <0.3：差相关性，0.3

表9流式细胞术的调查肿瘤中的DNA倍性

二倍体非整倍体

Table 9 Survey of flow cytometric DNA ploidy in the tumours

WHO grade Diploid Aneuploid Dipl S-phase, Aneup S-phase, Survival

mean mean Low High

7 1

3 16

10.7 25.93

16.0

8

19.55 8

Discussion

In the present tumour analyses there were fundamental changes in the cellular clockwork, both as estimated by their gene expression patterns and byimmunohistochemistry. The latter parameter not only showed quantitative changes in the tumour cells, but also alterations in the distributionbetween the nuclei and cytoplasm (Table 3). Several clock genes showed a down-regulation when compared to their own neighbouring mucosa, i.e. PER1, PER2 and PER3, while CRY2 was down-regulated in both tumour and neighbouring tissue when compared to normal mucosa from unrelated donors (Fig. 2a). In contrast, PER1 and CRY1 were up-regulated in tumour and neighbouring mu- cosa compared to the normal donor tissue. These findings were consistent with the IHC data (Table 3). One of the casein kinases (CSNK1A1), which is known to have acrit- ical regulatory role in transmitting signals from the clock genes, was reduced[27]. 在本发明的肿瘤分析中，细胞钟表发生了根本的变化，通过它们的基因表达模式和通过免疫组织化学来估计。后一个参数不仅显示肿瘤细胞的定量变化，而且还显示核和细胞质之间分布的改变（表3）。当与来自不相关供体的正常粘膜（图2a）相比时，当与它们自己的相邻粘膜，即PER1，PER2和PER3相比时，几个时钟基因显示下调，而在肿瘤和邻近组织中CRY2下调。相比之下，与正常供体组织相比，PER1和CRY1在肿瘤和相邻的mua cosa中上调。这些发现与IHC数据一致（表3）。已知在传输来自时钟基因的信号中具有关键调节作用的酪蛋白激酶（CSNK1A1）之一减少[27]。

We also found a moderately strong correlation be- tween the T/B ratios of PER2, CSNK1E and CSNK1A,re- spectively (Table 6). When using the neighbouring mucosa as reference to tumour, the picture became complex, since the mucosa may already have acquired preneoplastic properties or different influences from ma- lignant tissue. It was striking that clock gene aberrations were found mainly in aneuploid tumours of high grade. The same applied to increasing heterogeneity Litlekalsoyin tumour as well as neighbouringmucosa. etal.BMCCancer(2016)16:549

我们还发现PER2，CSNK1E和CSNK1A的T / B比率之间具有适度强的相关性（表6）。当使用相邻的粘膜作为肿瘤的参考时，图片变得复杂，因为粘膜可能已经获得了肿瘤前性质或来自木本组织的不同影响。令人惊讶的是，时钟基因畸变主要发现在高等级的非整倍体肿瘤中。这同样适用于增加肿瘤以及相邻粘膜的异质性。

When compared to normal unrelated mucosa, all the cancer related oncogenes except HRAS, were strongly up-regulated, while the two suppressor genes TP53 andPTEN were down-regulated (Fig. 2b). In line with other studies [28], we have earlier reported that there is an accu- mulation of the p53 protein in these tumour cells, possibly a non-functional suppressor protein, while PTEN seemsto be largely absent [25, 29]. The strong up-regulation of high molecular weight cytokeratins found in the gene- expression analysis is also consistent with our earlier find- ings [25]. The accumulation of these proteins has been related to a worse prognosis. The same relates to an up- regulation of the plasminogen activator (uPAR) and the inhibitor, PAI-1, which at high levels paradoxically stimulatesinvasivegrowth. 与正常无关的粘膜相比，所有癌症相关癌基因，除了HRAS，都被强烈上调，而两个抑制基因TP53和PTEN被下调（图2b）。与其他研究[28]一致，我们早先报道了在这些肿瘤细胞中存在p53蛋白的积累，可能是非功能性抑制蛋白，而PTEN似乎基本上不存在[25,29]。在基因表达分析中发现的高分子量细胞角蛋白的强上调也与我们早先的发现一致[25]。这些蛋白质的积累与更差的预后有关。这同样涉及纤溶酶原激活物（uPAR）和抑制剂PAI-1的上调，其在高水平上矛盾地刺激侵袭性生长。 PAI-1 expression in different tissues is closely con- trolled by clock genes in vivo. Loss of clock genes may result in an increased PAI-1 expression and constitutes a contributing risk factor for cardiovascular disease. There is also a possibility that CRY suppresses PAI-1 expres- sion independent of its clock function. It has been sug- gested that clock genes and RAS may differentially affect the circadian expression of PAI-1 in various tissues. Other studies reveal that the basic helix-loop-helix (bHLH)/PAS domain transcription factor plays a crucial role in control- ling the biological clock that control thecircadian rhythms. In line with this, a novel bHLH/PAS protein cycle-like factor (CLIF) regulates the circadian regulation ofPAI-1geneexpressioninendothelialcells[30,31]. 不同组织中PAI-1的表达受体内时钟基因的紧密控制。时钟基因的丢失可能导致PAI-1表达增加，并构成心血管疾病的危险因素。也有可能CRY抑制PAI-1表达独立于其时钟功能。已经提出时钟基因和RAS可以差异地影响PAI-1在各种组织中的昼夜表达。其他研究表明基本螺旋 - 环 - 螺旋（bHLH）/ PAS域转录因子在控制生物钟控制昼夜节律中发挥关键作用。与此相一致，一种新的bHLH / PAS蛋白质循环样因子（CLIF）调节内皮细胞中PAI-1基因表达的昼夜节律调节[30,31]。

A surprising finding was that oncogene overexpres- sion Page 17of 17

was both correlated to the levels of stimulatoryand inhibitory clock genes (Table 8). We have earlier reported a strong up-regulation of EGFR and p16, in- cluding H-, K- andN-RAS [25,29],andinthe presentmRNAanalysisallofthesetumourgenes were strongly correlated (Table 7 and 8). This extends earlier findings that malignant behaviour in urothelial cells may at least in part be due to a combined action of oncogenes, altered suppressor genes and aberrantclock gene expression [32–34]. However, the presentdata donotgiveanyinformationwithregardsto which of these three gene classes is the primary cause of this deviation. Alternatively, mutations and/or deletions in either of them, leading to non-functional proteins could becriticalstepsindevelopmentofbiologicalmalignancy. 令人惊讶的发现是致癌基因过表达与刺激和抑制性时钟基因的水平相关（表8）。我们早先报道了EGFR和p16的强上调，包括H-，K-和N-RAS [25,29]，在目前的mRNA分析中，所有这些肿瘤基因都是强相关的（表7和 8）。这扩展更早的发现，泌尿上皮细胞的恶性行为可能至少部分是由于癌基因，改变抑制基因和异常时钟基因表达的组合行动[32-34]。然而，本数据没有给出关于这三种基因类型中哪一种是这种偏差的主要原因的任何信息。或者，其中任一种突变和/或缺失导致非功能性蛋白质可能是生物恶性肿瘤发展中的关键步骤。

The finding that such combined aberrations are almost exclusively in high grade, aneuploid tumours, points in the same direction. Thus, it has been known for several decades that aneuploid urothelial cancers have a higher malignant potential, accompanied by a higher frequency of aneuploidy in the neighbouring normal appearing mucosa [26]. The ex- pressions of uPAR and PAI-1, which mediate a cascade of other cellular functions related to invasiveness and prote- olysis, were also correlated to alterations of the clock gene expression points in the same direction (see Table 8). 这种组合的像差几乎仅在高级，非整倍体肿瘤中的发现指向相同的方向。因此，已知几十年来，非整倍体尿路上皮癌具有较高的恶性潜能，伴随着在邻近正常出现的粘膜中更高的非整倍性频率[26]。 uPAR和PAI-1的表达，其介导与侵袭和蛋白水解相关的其他细胞功能的级联，也与时钟基因表达点在相同方向上的改变相关（参见表8）。

In rodents, it has been reported that mutation of theCSNK1 priming site in PER2 (Ser662), leads to decreasedphosphorylation of stabilizing sites in PER2 and acceler- ated circadian rhythms. PER1 and 2 have the highest amp- litude oscillations of all the known core clock proteins, with almost complete degradation near the end of the subjective night in the SCN. PER2 also undergoes tem- poral changes in phosphorylation that reaches a zenith just prior to its destruction. Both kinase/phosphatase ac- tivities are thought to regulate PER2 net phosphorylation and stability [35–37]. PER2 has also been found to func- tion as a tumour suppressor, with the absence of both its copies causing an increased rate of radiation-induced can- cers. It now seems evident that its anti-cancer action arises from the ability to turn off Myc. In the absence of PER2, Myc levels greatly rise, thereby explaining why many types of tumours display higher levels of CSNK1E than their normal cell equivalents[38]. Litlekalsoy在啮齿类动物中，据报道，etal.BMCCancer(2016)16:549

PER2（Ser662）中CSNK1引发位点的突变导致PER2中稳定位点的磷酸化减少，并加速昼夜节律。 PER1和2具有所有已知核心时钟蛋白的最高电位振荡，在SCN主观夜晚结束附近几乎完全降解。 PER2也经历了磷酸化的时间变化，其在破坏之前达到天顶。激酶/磷酸酶活性被认为调节PER2净磷酸化和稳定性[35-37]。 PER2也被发现作为肿瘤抑制基因，其两个拷贝的缺乏导致辐射诱导的癌症的增加的速率。现在似乎很明显，其抗癌作用起因于关闭Myc的能力。在不存在PER2的情况下，Myc水平显着升高，从而解释了为什么许多类型的肿瘤显示比其正常细胞等同物更高水平的CSNK1E [38]。 The majority of all advanced human tumours have mutations in the TP53 gene, and in rodents PER2 expression is also found directly regulated by p53 bind- ing to a response element in the PER2 promoter. This p53 response element is evolutionarily conserved and overlaps with the E-Box element critical for BMAL1/ CLOCK binding and its transcriptional activation of PER 2 expression. In consequence, p53 may block BMAL1/ CLOCK binding to the PER2 promoter, where the cellu- lar level of PER2 is inversely correlated with that of p53. Studies also suggest that functional PER2 is important for p53-mediated stress signals to reach the circadian clock network and that p53 acts as a transcription factor that regulates the circadian clock by direct control of PER2 expression [39]. A common paradox is that that there may be an accumulation of the protein in malig-nant cells in spite of their unrestricted growth. Our ob- servation of down-regulation of the gene expression combined with accumulation of p53 in urothelial cancer (Fig. 2) is therefore a common finding in these tumours [25, 40]. Surprisingly, the reduced transcription of the tumour suppressor p53 was correlated to the expression of clock genes and related casein kinases (Table 8). 所有晚期人类肿瘤中的大多数在TP53基因中具有突变，并且在啮齿动物中，也发现PER2表达直接受p53结合PER2启动子中的响应元件调节。该p53应答元件在进化上保守并且与对于BMAL1 / CLOCK结合及其PER 2表达的转录激活关键的E-Box元件重叠。因此，p53可阻断BMAL1 / CLOCK与PER2启动子的结合，其中PER2的细胞水平与p53的细胞水平负相关。研究还表明功能性PER2对于p53介导的应激信号到达昼夜节律钟网络是重要的，并且p53充当通过直接控制PER2表达调节昼夜节律钟的转录因子[39]。一个常见的悖论是，尽管其无限制生长，可能在恶性细胞中存在蛋白质的积累。我们观察到的基因表达的下调以及p53在尿路上皮癌中的累积（图2）因此是这些肿瘤中的常见发现[25,40]。令人惊讶的是，肿瘤抑制基因p53的转录减少与时钟基因和相关的酪蛋白激酶的表达相关（表8）。 Since we have only investigated cystectomies and un- related normal mucosa harvested in the first part of the light period, i.e. before noon, high transcriptional levels of the inhibitory genes and low levels of the Page 18of 17stimulatory

ones would be expected. As shown in our Results, thiswas not the case, indicating a disturbance of the circa- dian timing in the malignant urothelial cells. However,two open questions remain: Could the observed clock gene alterations be due to a longstanding phase shift of otherwise normal oscillations and not a disruption of the clock work per se? Although our data do not warrant a firm conclusion on these questions, they mainly suggesta severe perturbation of the cellular clocks. One can speculate whether the preparation for surgery and sur- gery/anesthesia itself might lead to differential disruptionof endogenous circadian homeostasis in both normaland tumour tissue. However, all the patients included in our study are diagnosed with bladder cancer and have undergone the same surgical procedure. 因为我们仅研究在光周期的第一部分即中午之前收获的囊肿切除术和不相关的正常粘膜，所以将期望抑制性基因的高转录水平和低水平的刺激性基因。如我们的结果所示，这不是这种情况，表明恶性泌尿道上皮细胞中的周期时间的干扰。然而，两个开放的问题仍然存在：观察时钟基因改变是否是由于其他正常振荡的长期相移，而不是时钟工作本身的中断？虽然我们的数据不保证对这些问题的确定的结论，他们主要建议蜂窝时钟的严重扰动。可以推测手术和手术/麻醉的准备本身是否会导致正常组织和肿瘤组织内源性昼夜节律内稳态的差异破坏。然而，包括在我们的研究中的所有患者被诊断为膀胱癌并且经历了相同的外科手术。 Corresponding studies of clock genes in human tissues used for com- parison are also conducted on tissue harvested from sur- gical specimens. The close relation of these changes tothe up-regulation of other cancer-associated genes also indicates a disruption of the clock. Since sequence sam- pling of cancerous tissue fragments and cells for investi- gating the whole circadian cycle is at present notclinically possible, a final answer remainsopen. 对用于比较的人体组织中的时钟基因的相应研究也对从外科标本收获的组织进行。这些变化与其他癌症相关基因的上调的密切关系也指示时钟的破坏。由于癌症组织片段和细胞的用于研究整个昼夜节律循环的序列取样目前在临床上是不可能的，所以最终答案仍然是开放的。

Since biological markers for bladder cancer reported so far have only been of limited clinical value [32], clock gene markers might therefore serve as an adjunct toother diagnostic and prognostic histological/biological markers. The present study has some limitations with respect to the number of cases included, which hence af- fects the statistical power and makes it difficult to draw a broad conclusion. However, the strong significance be- tween several independent parameters makes it unlikely that this is due to random variations. Future expanded studies are warranted to validate the role of the different markers selected in this study.

因为到目前为止报道的用于膀胱癌的生物标志物仅具有有限的临床价值[32]，因此时钟基因标志物可以作为其他诊断和预后组织学/生物标志物的辅助。本研究对所包括的病例数有一些限制，从而影响统计学的力量，难以得出广泛的结论。然而，几个独立参数之间的重要意义使得这

Litlekalsoyetal.BMCCancer(2016)16:549

不可能是由于随机变化。未来扩展的研究保证验证本研究中选择的不同标记的作用。

Conclusions

A correlation was found between altered mRNA and protein expression of various clock genes and common tumour markers in urothelial cancer, indicating that dis- turbed function in the cellular clockwork may be an im- portant additional mechanism contributing to cancer progression and malignant behaviour. These alterations

are most pronounced in aneuploid, high grade tumours, and are to some extent also seen in the neighbouring mucosa.

TLDA，taqman低密度阵列; TUR-P，经尿道前列腺切除术; V.I.，血管浸润

Page 19of 17

Acknowledgements致谢

We thank Ms. Anne Aarsand for technical assistance with the

immunohistochemical analyses, Ms. Beth Johannessen for guidance and assistance with the gene-expression studies and professor August Bakke for valuable advice. We are also very grateful to Dr. Hawa Nalwoga for her support with statistical calculations.

我们感谢Anne Aarsand女士提供技术援助

免疫组化分析，Beth Johannessen女士对基因表达研究的指导和帮助，以及August Bakke教授的宝贵意见。我们还非常感谢Hawa Nalwoga博士对统计计算的支持。

Funding

The project was supported by the Uro-Bergen Fund and the Norwegian Cancer Society.该项目得到了欧洲 - 卑尔根基金和挪威癌症协会的支持。 在尿路上皮癌中，发现改变的mRNA和各种时钟基因和常见肿瘤标志物的蛋白质表达之间的相关性，表明细胞钟表中的混乱功能可能是导致癌症进展和恶性行为的重要的另外的机制。这些改变在非整倍体，高级肿瘤中最显着，并且在一定程度上也在相邻粘膜中见到。

Additional file

Additionalfile1:FigureS1.Immunohistochemicalstainingofvarious clockproteinsinurothelialcancerascomparedtonormalcontrolsstainedin parallel.Counterstainedwithhematoxylin(bluenuclei).Magnificationisgiven bythetoolbar:10μm.A:PER1intumourshowingthesamestainingdensity as in controls (B). C: PER3 with positive nuclei in tumour as compared to negativeinthecontrols(D).Thecytoplasmisslightlypositiveinboth.E:CRY1 with increased positivity in tumour cell cytoplasm as compared to the control (F). G: CRY2 with negative reaction in tumour cell nuclei and positive in controls (H). I: BMAL-1 with approximately equal staining in tumour cells and control (J), i.e. moderate staining in nuclei and strong in cytoplasm. For semiquantitativeestimatesanddetails,seeTable3.(PDF617kb) 附加文件1：图S1。与正常对照平行染色相比，尿路上皮癌中各种时钟蛋白的免疫组织化学染色。用苏木精（蓝色核）复染色。放大倍率由工具条给出：10μm。 A：肿瘤中的PER1显示与对照相同的染色密度（B）。 C：与对照中的阴性相比，肿瘤中具有阳性核的PER3（D）。细胞质在两者中都是阳性的。 E：与对照（F）相比，肿瘤细胞质中阳性增

加的CRY1。 G：CRY2，在肿瘤细胞核中具有阴性反应，在对照中为阳性（H）。 I：BMAL-1在肿瘤细胞和对照（J）中具有大致相等的染色，即细胞核中的中度染色和细胞质中的强染色。对于半定量估计和细节，见表3.（PDF 617 kb）

Abbreviations缩写

A, aneuploid; A-S, Aneuploid S-phase; B, benign tissue; bHLH, basic helix- loop-helix;BPH,benignprostatichyperplasia;C,correlationcoefficient;CLIF, cycle-like factor; D, diploid; D-S, Diploid S-phase; FCM, flow cytometry; G, grade;N,normaltissue;PI,ploidyindex;qPCR,quantitativepolymerasechain reaction;r,Pearson’sproductmonumentcorrelationcoefficients;RQ,relative quantity;SEM,standarderrorofmean;SurvivalA/D,survivalaftersurgery;T,tumour tissue; TLDA, taqman low density arrays; TUR-P, transurethral resection of the prostate; V.I., vascular invasion A，非整倍体; A-S，非整倍体S期; B，良性组织; bHLH，碱性螺旋 - 环 - 螺旋; BPH，良性前列腺增生; C，相关系数; CLIF，周期样因子; D，二倍体; D-S，二倍体S期; FCM，流式细胞术; G，等级; N，正常组织; PI，倍性指数; qPCR，定量聚合酶链反应; r，Pearson的积相关系数; RQ，相对量; SEM，平均值的标准误差; 生存A / D，术后生存; T，肿瘤组织;

Availability of data and materials数据和材料的可用性

The dataset supporting the conclusions of this article is included within the article (and its additional file).

Authors’ contributions

JL and ODL conceived and initiated the study. JL, KR and JGH performed the experiments. JL, KR, JGH, ODL participated in the study design, data analysis and interpretation. JL, KR and ODL drafted the manuscript. KHK participated in the molecular analysis and critical revision of the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests. Consent for publication Not applicable.

Ethics approval and consent to participate

Patients’ informed consent was obtained for all human biopsies utilized in this study. Approval was attained by the Regional Ethical Committee

(“Regionale komiteer for medisinsk og helsefaglig forskningsetikk”; REK No. 12226/REK No. 2009/1527).

Author details 1

Department 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. 3Institute of BiomedicalLaboratorySciencesandChemicalEngineering,BergenUniversity College,Bergen,Norway.4DepartmentofMicrobiology,HaukelandUniversity Hospital, Bergen, Norway. 5Department of Urology, Surgical Clinic, Haukeland University Hospital, Bergen, Norway. 6Department of Pathology, Haukeland University Hospital, Bergen, Norway. 7Department of Clinical Science, Haukeland University Hospital, N-5021 Bergen, Norway. Received: 12 October 2015 Accepted: 19 July 2016

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