Expression of circadian clock genes and(2)

Litlekalsoyetal.BMCCancer(2016)16:549

Endogenous control and endogenous control cards内生控制和内在控制卡

The different tissue types included in our study were initially studied with respect to gene expression of 16 different housekeeping genes, to assess which one was best suited as endogenous control for our purpose. Two endogenous control cards accommodating 8 samples each, in triplicate, were applied. β-actin (ACTB) proved to be the most suitable endogenous control for our three tissue types and therefore chosen when designing the Taqman low density arrays

Antigen抗原 PER1 (Per12-A) PER2 (N-19, sc-7728) PER3 (Per32-A) CRY1 (W-L5, sc-101006) CRY2 (P-21, sc-130731) BMAL 1 (LS-B660/12275) CLOCK (LS-B278/18928 Anti-CSNK1α1L

Specificities Purchaser买方 Polyclonal多克隆 Polyclonal Polyclonal

AH Diagnostics AS Fjellgata 1, Oslo

(TLDA) cards. In addition GAPDH was added in the TLDA cards as standard (from the supplier), but was not used in our further calculations. 最初研究包括在我们的研究中的不同组织类型关于16个不同管家基因的基因表达，以评估哪一种最适合作为内源对照用于我们的目的。应用两个内部控制卡，每个容纳8个样品，一式三份。β-肌动蛋白（ACTB）被证明是最适合我们的三种组织类型的内源性对照，因此在设计Taqman低密度阵列（TLDA）卡时被选择。此外，GAPDH作为标准（从供应商）加入TLDA卡中，但不用于我们的进一步计算。

Page 5of 17

Table 2 Specifications of antigens and corresponding antibodies抗原和相应抗体的规格

Dilution稀释

1:50, overnight at 4 °C

Pre-treatment预处理

Microwave treatment for 10 min at 750 W and 20 min at 500 W in 10 mmol/L citrate buffer pH6 Microwave treatment for 10 min at 750 W and 20 min at 500 W in 10 mmol/L citrate buffer pH6 Microwave treatment for 10 min at 750 W and 20 min at 500 W in 10 mmol/L citrate buffer pH6 Microwave treatment for 10 min at 750 W and 20 min at 500 W in 10 mmol/L citrate buffer pH6 Microwave treatment for 10 min at 750 W and 20 min at 500 W in 10 mmol/L citrate buffer pH6 Microwave treatment for 10 min at 750 W and 20 min at 500 W in 10 mmol/L citrate buffer pH6 Microwave treatment for 10 min at 750 W and 20 min at 500 W in 10 mmol/L citrate buffer pH6 Microwave treatment for 10 min at 750 W and 20 min at 500 W in 10 mmol/L citrate buffer pH6 Microwave treatment for 10 min at 750 W and 20 min at 500 W in 10 mmol/L citrate buffer pH6 Microwave treatment for 10 min at 750 W and 20 min at 500 W in 10 mmol/L citrate buffer pH6

Santa Kruz Biotecnology 1:200, overnight at 4 °C Inc. Europe AH Diagnostics AS Fjellgata 1, Oslo

1:50, overnight at 4 °C

MonoclonalSanta Kruz Biotecnology 1:200, overnight at 4 °C 单克隆 Inc. Europe Polyclonal Polyclonal Polyclonal Polyclonal

Santa Kruz Biotecnology 1:200, overnight at 4 °C Inc. Europe Lifespan Biosciences (Nordic biosite) Lifespan Biosciences (Nordic biosite) Abcam.com England

1:100, overnight at 4 °C 1:500, overnight at 4 °C 1:150, overnight at 4 °C

Casein kinase 1? (Sc-25423) Polyclonal Casein kinase 1α (Sc-28886) Polyclonal

Santa Kruz Biotecnology 1:100, overnight at 4 °C Inc. Europe

Santa Kruz Biotecnology 1:100, overnight at 4 °C Inc. Europe

在10mmol / L柠檬酸盐缓冲液pH6中在750W微波处理10分钟和在500W微波处理20分钟

Litlekalsoyetal.BMCCancer(2016)16:549

Table 3Mean scores of positivity in nucleus and cytoplasm for the clockproteins时钟蛋白的核和细胞质中的阳性的平均得分

蛋白癌细胞临近粘膜正常粘膜 细胞核细胞质

Protein Cancercells Neighbouring mucosa Normalmucosa

Nucleus Cytopl. Nucleus

Cytopl. Nucleus Cytopl. PER 1 2.17 0 1.71 0 2.00 0 +/? SEM 0.15 0 0.10 0 0 0 PER 3 0.22 0.43 0.67 0.73 0 1.15 +/? SEM 0.08 0.11 0.13 0.15 0 0.13 CRY 1 2.08 1.96 1.84 1.27 1.76 0.62 +/? SEM 0.16 0.11 0.14 0.20 0.13 0.15 CRY 2 0 0.83 2.31 2.75 2.00 2.00 +/? SEM 0 0.13 0.23 0.18 0.26 0.28 BMAL1 1.42 2.40 2.33 2.27 1.16 2.08 +/? SEM 0.20 0.12 0.19 0.20 0.21 0.20 CLOCK

2.04

2.23 2.57 2.52 2.75 2.91 +/? SEM 0.16

0.12 0.13 0.13

0.14 0.09 Casein kinase 1 alpha

1.93

2.70 2.78 3.00 2.90 3.00 +/? SEM 0.18

0.10 0.13 0.00 0.11 0.00 Casein kinase 1 alpha 1 L

1.96

2.59 2.88 2.96 2.54 2.92 +/?SEM 0.24

0.12 0.08 0.04 0.22 0.08 Casein kinase 1 epsilon

2.93

2.07 2.75 1.95 3.00 2.00

+/? SEM 0.05

0.05

0.10

0.09

0

0

+/? SEM: +/? standard error of the arithmetic means算术平均值的标准误差

Page 6of 17

Real-time quantitative PCR (qPCR) in low-density array format实时定量PCR（qPCR）在低密度阵列格式

Taqman low density arrays (TLDA) are customizable,384-well microfluidic cards for real-time qPCR (AppliedBiosystems (ABI)). Each TLDA card was configuredfor 24 genes in duplicates, including β-actinand GAPDHasendogenouscontrols, coreclock-genes and genes encoding several tumour markers (TaqMan assays are listed in Table 4). Single-stranded cDNA corresponding to 200 ng of total RNA was diluted in Taqman Universal buffer (ABI) and added to eachloading well. The samples were distributed to the mi- cro wells by centrifugation for 1 min at 343xg. Thecards were placed in an ABI PRISM 7900HT Se-quence Detection System thermocycler for 40 cycles:15 s at 95 °C and 60 s at 60 °C. The SDS2.3 and RQ manager 1.2 software (ABI)were usedforanalysis and data were exported to Excel for further visuali-zation. Data Assist v.3.01 (ABI) was utilized for hier- archical cluster analysis and generation of correlation plots. The gene expression data were analysed usingthe comparative Ct-method (ΔΔCt). Gene expression levels were normalized against ?-actin and calibrated against a chosen calibrator toprovidefoldchange relative gene expression levels. Two separate gene ex- pressionanalysiswere performed in ordertostudy the relative differential gene expression (fold change (Relative quantity (RQ)) in the respective tissues: tumour and neighbouring benign tissue relative to unrelated nor- mal mucosa, and relative gene expression levels in tumour versus neighbouringmucosa.

Taqman低密度阵列（TLDA）是可定制的384孔微流体卡，用于实时qPCR（Applied Biosystems（ABI））。每个TLDA卡被配置为一式两份的24个基因，包括作为内源对照的β-肌动蛋白和GAPDH，核心时钟基因和编码几种肿瘤标记物的基因（TaqMan测定列于表4）。将对应于200ng总RNA的单链cDNA在Taqman通用缓冲液（ABI）中稀释并加入到每个加样孔中。通过在343xg下离心1分钟将样品分配到微量孔中。将卡放置在ABI PRISM 7900HT序列检测系统热循环仪中进行40个循环：95℃15秒和60℃60秒。 SDS2.3和RQ管理1.2软件（ABI）用于分析，数据输出到Excel进一步可视化。数据辅助v.3.01（ABI）用于层次聚类分析和相关图的生成。使用比较Ct法（ΔΔCt）分析基因表达数据。将基因表达水平针对β-肌动蛋白标准化，并针对选择的校准物校准以提供倍数变化相对基因表达水平。进行两个单独的基因表达分析，以研究相对差异基因表达（倍数变化（相对量（RQ））在相应的组织：肿瘤和邻近良性组织相对于无关的正常粘膜和相对基因表达肿瘤相对于邻近粘膜的水平

Litlekalsoyetal.BMCCancer(2016)16:549 Page 7of 17

Table 4 List of TaqMan gene expression assays and their corresponding proteins

Gene assay Hs00978050_m1 Hs00364284_m1 Hs00180035_m1 Hs01076078_m1 Hs00182181_m1 Hs01126606_m1

Protein H-RAS K-RAS N-RAS EGFR uPAR PAI 1

Gene assay Hs01034249_m1 Hs00923894_m1 Hs02621230_m1 Hs00559840_m1 Hs00196158_m1 Hs00361185_m1 Hs00166289_m1

GAPDH

Hs00265033_m1

Protein p53 p16 pTEN Cytokeratin 7 Cytokeratin 1 Cytokeratin 5 Cytokeratin10 Cytokeratin14

Gene assay Hs00242988_m1 Hs00256143_m1 Hs00213466_m1 Hs01565974_m1 Hs00323654_m1 Hs00154147_m1 Hs00231857_m1 Hs01887794_m1 Hs00793391_m1 Hs00266431_m1

Protein PER 1 PER 2 PER 3 CRY 1 CRY 2 BMAL 1 CLOCK CK1A1L CK1A1 CK1ε

Hs99999905_m1

Statistics统计

Statistical Package for the Social Sciences (SPSS v.12) (SPSS Inc. Chicago, Illinois) was utilized for statisticalanalysis. The Spearman’s rank correlation (correlationsco- efficient, c) was used to determine significant correlation between the various gene expressions. The Mann- Whitney non-parametric rank test was used to identify correlation between the gene expressions in the tumours compared to neighbouring mucosa. Data Assist v.3.01 (ABI) was applied on the gene expression data tocalculate Pearson’s product monument correlation coefficients (r) for each sample represented in the various tissue types. Pearson’s correlation was used for the hierarchical cluster analysis and generation of heat maps of gene expression. Data Assist v.3.01 (ABI) performed a two-sample, two- tailed Student’s t-test for comparing the fold (?deltaCt)changevalues(2) of the separate biological groups (normal bladder mucosa, neighbouring benign and tumour tissue), and a p-value was calculated. The results were presented inthe mRNA fold change gene expression plots (log fold changeversussamplegroup). 社会科学统计包（SPSS v.12）（SPSS Inc.，Chicago，Illinois）用于统计分析。使用斯皮尔曼秩相关（相关系数，c）来确定各种基因表达之间的显着相关性。使用Mann-Whitney非参数秩检验来鉴定肿瘤中的基因表达与相邻粘膜的相关性。将数据辅助v.3.01（ABI）应用于基因表达数据以计算在各种组织类型中表示的每个样品的皮尔逊积积相关系数（r）。 Pearson相关性用于层次聚类分析和基因表达热图的产生。数据辅助v.3.01（ABI）进行了两个样本的双尾学生t检验，用于比较单独的生物组（正常膀胱粘膜，邻近的良性和肿瘤组织）的倍数变化值（2（-deltaCt），并计算p值。结果以mRNA倍数变化基因表达图（对数倍变化对样品组）表示。

was significantly increased in neighbouring tissue, and also slightly increased in tumour tissue compared to normal mucosal cells (Table 3). Six cases expressed nei- ther BMAL1 nor CRY2 in the nucleus. When this was compensated for, the remaining positive cases for BMAL1 had a mean score in the nucleus of 1.84 +/? SEM 0.15, which is significantly higher than in the nor- mal mucosa. CLOCK was significantly reduced in the tumour cells, but not in the nucleus or cytoplasm in the neighbouringmucosa. 细胞质BMAL1染色在肿瘤和相邻粘膜细胞中比在正常的，不相关的粘膜中稍强。在核中，BMAL1在相邻组织中显着增加，并且与正常粘膜细胞相比在肿瘤组织中略微增加（表3）。 6例在细胞核中表达为BMAL1或CRY2。当这被补偿时，BMAL1的剩余阳性病例在核中具有1.84 +/- SEM 0.15的平均得分，其显着高于正常粘膜。 CLOCK在肿瘤细胞中显着减少，但在相邻粘膜的细胞核或细胞质中不显着。

Casein kinase 1A and 1A1Like were both significantly reduced in the tumour nuclei, but not in the cytoplasm. Casein kinase 1E was equally expressed in both nucleus and cytoplasm. 酪蛋白激酶1A和1A1像我们在肿瘤细胞核中显着减少，但不是在细胞质中。酪蛋白激酶1E在核和细胞质中同样表达。

Results

Immunohistochemistry免疫组织化学

Stimulatory clock proteins/casein kinases刺激时钟蛋白/酪蛋白激酶

Cytoplasmic BMAL1 staining was slightly stronger in the tumour and the neighbouring mucosal cells than in the normal, unrelated mucosa. In the nuclei, BMAL1 Litlekalsoyetal.BMCCancer(2016)16:549

Inhibitory clock proteins抑制时钟蛋白

PER1 was positive in the nucleus and absent in cyto- plasm of neoplastic, neighbouring and normal mucosa (Table 3). PER2 did not give satisfactory staining and was omitted. PER3 was absent in nucleus of normal mu- cosa, but expressed in cancer cells and their neighbour-ing mucosa. Opposite, it was lower in the cytoplasm of cancer cells and neighbouring tissue compared to nor- mal mucosa, and there seemed to be a significant shift

from cytoplasm to nucleus in malignancy. CRY1 was significantly increased in tumour cytoplasm and neigh- bouring mucosal cells.Theincreasedexpressionof CRY1 in the cancer cells was three times higher than in normal mucosa. CRY2 was absent in the nucleus in cancer cells and low in the cytoplasm, while neigh- bouring and normal mucosal cells showed no major differences. PER1在核中是阳性的，在肿瘤，邻近和正常粘膜的细胞质中不存在（表3）。 PER2没有得到令人满意的染色并省略。 PER3在正常mua cosa的核中缺失，但在癌细胞及其相邻粘膜中表达。相反，与正常粘膜相比，癌细胞和邻近组织的细胞质中更低，并且在恶性肿瘤中似乎有从细胞质到核的显着转变。 CRY1在肿瘤细胞质和邻近粘膜细胞中显着增加。癌细胞中CRY1的表达增加是正常粘膜中的3倍。 CRY2在癌细胞核中缺失，在细胞质中低，而邻近和正常粘膜细胞没有显着差异。

Altogether, this indicates complex alterations, where the main features were redistribution between nucleus and cytoplasm, and an increase ofboth stimulatoryand inhibitory clock proteins, see in Additional file 1: FigureS1. 总之，这表明复杂的变化，其中主要特征是核和细胞质之间的重新分配，以及刺激和抑制性时钟蛋白的增加，参见附加文件1：图S1。

肿瘤中mRNA的相对数量对于发现统计学显着的基因。图5显示了来自15个不相关供体的正常粘膜中22个基因与来自27个患者（囊肿切除术）的肿瘤/相邻粘膜的差异表达的层次聚类图（热图）。

Page 8of 17

Gene expression analysis基因表达分析

Raw data and general pattern原始数据和一般模式

The over-all differences in gene expression pattern in tumours compared to matched neighbouring mucosa are shown in Table 5. The gene-expression signal cor- relation plot is visualized in Fig. 1. The mRNA fold change in tumour and neighbouring mucosa from 27 patients relative to normal mucosa from 15 unrelated donors are visualized in Fig. 2. Figures 3 and 4 display relative quantity of mRNA in tumour compared to neighbouring mucosa of 27 patients for the genes found statistically significant. Figure 5 shows a hierarchical cluster diagram (heat map) of differential expression of 22genesinnormalmucosafrom15unrelateddonorsto- gether with tumour/neighbouring mucosa from 27 pa- tients(cystectomies). 与匹配的相邻粘膜相比，肿瘤中基因表达模式的总体差异显示在表5中。基因表达信号相关图在图5中可视化。来自17个患者的肿瘤和相邻粘膜相对于来自15个不相关供体的正常粘膜的mRNA倍数变化在图1中可视化。图3和4显示了与27名患者的相邻粘膜相比，

表5来自囊肿切除术的时钟基因和常见肿瘤标志物的相对基因表达水平（肿瘤/良性倍数变化）

A.来自囊肿切除术的时钟基因和常见肿瘤标志物的相对mRNA基因表达水平（肿瘤/良性倍数变化）

Table 5 Relative gene expression levels of clock genes and common tumour markers from cystectomies (Tumour/Benign-fold change)

A. RelativemRNAgeneexpressionlevelsofclockgenesandcommontumourmarkersfromcystectomies(Tumour/Benign-foldchange) GENES Patient sample 1 2 BMAL CLOCK PER1 PER2 PER3 CRY1 CRY2 CSNK1A1L CSNK1A1 CSNK1E TP53 p16 1,3 2,1 0,8 1,0 0,1 0,9 0,1 1,2 0,3 0,8 0,5 1,5 0,3 1,3 34,8 0,0 0,6 1,3 0,5 3,5 0,7 2,0 8,1 6,3 PTEN EGFR HRAS KRAS NRAS Upar PAI-1 KRT7 KRT1 KRT5 KRT10 KRT14 0,5 2,4 0,8 0,8 0,9 1,9 0,6 1,0 0,9 2,3 0,1 1,6 0,1 0,6 0,5 83* 1,1 0,0 0,0 0,3 0,1 0,1 9,5 311* Litlekalsoyetal.BMCCancer3 4,9 4 1,0 5 1,3 6 3,6 7 1,4 8 2,8 9 0,5 10 0,6 11 1,5 12 4,3 13 4,1 14 1,1 15 3,6 16 0,8 17 2,3 18 1,9 19 0,8 20 2,0 21 0,5 22 0,8 23 0,5 24 1,2 25 2,6 26 1,0 27

1,0

3,2 0,4 1,1 0,2 1,2 0,5 1,6 1,4 2,3 0,9 2,1 0,0 1,3 1,7 0,3 0,3 1,3 0,6 1,7 0,8 1,0 2,0 0,3 0,1 2,2 0,3 0,7 0,3 1,2 0,2 1,8 0,2 0,4 0,3 2,8 0,6 1,2 0,7 0,8 0,7 1,3 1,7 1,1 0,4 1,1 0,5 0,7 0,5 0,7

0,1

0,7 5,9 3,9 0,3 0,6 0,4 0,3 0,8 0,7 0,5 0,7 2,3 1,0 0,9 2,4 0,6 1,6 2,8 0,4 5,9 1,0 0,2 0,8 1,4 1,0 1,0 0,9 0,4 0,6 0,6 1,7 0,5 1,3 0,3 0,4 0,0 0,4 1,6 1,4 0,4 0,4 0,6 0,5 1,3 2,0 0,2 0,2 1,5 0,3 0,3 0,7 0,3 0,9 0,6 0,5 0,7 1,5 0,3 0,1 1,0 0,4 5,9 1,0 0,7 0,3 0,3 0,6 1,3 0,5 1,5 0,2 0,7 1,0

0,0

0,4

0,8 3,2 0,7 37,1 0,5 0,1 1,2 177 1,0 0,0 3,1 6,6 2,2 0,2 0,6 169 1,1 0,3 0,4 0,0 1,5 1,5 0,1 0,0 1,3 6,2 0,6 0,6 0,9 0,1 0,2 1,2 0,6 0,6 0,8 0,0 1,0 0,4 0,6 0,0 2,2 0,2 0,3 1,1 0,5 21,9 0,4 0,0 0,2

27,9

3,2 11,3 0,6 0,3 0,4 0,5 1,3 3,0 1,3 7,4 1,9 8,5 0,7 0,4 0,5 0,4 1,0 1,2 1,5 1,9 0,9 0,5 0,2 0,2 1,3 1,8 1,8 0,8 0,8 0,9 1,1 1,0 0,7 0,5 1,0 1,0 0,8 0,7 1,2 2,4 0,7 0,4 1,1 0,8 1,2 1,7 1,7 1,0 2,5

2,3

0,9 131 1,9 8,7 2,2 0,9 0,7 1,5 0,8 0,6 0,7 0,5 2,9 88,0 0,8 1,0 1,9 1,1 0,7 1,1 1,8 12,9 39,0 2,2 0,6 0,2 0,4 0,8 1,3 0,0 1,1 3,0 3,4 24,0 1,2

1,7 3,6 1,3 4,0 1,1 2,8 3,9 5,7 0,6 0,4 2,6 1,9 0,1 4,0 7,9 2,6 2,0 0,9 0,8 0,8 1,2 3,3 1,9

0,8

3,8 3,0 84,0 0,7 3,4 1,2 10,0 1,1 4,2 3,4 1,9 1,0 0,6 1,9 1,3 0,9 2,1 4,3 4,2 1,9 2,8 0,6 0,2 0,4 0,6 1,4 0,3 0,9 1,7 1,6 1,7 1,1 0,8 1,3 1,2 4,7 0,4 3,8

0,7

7,6

1,4

8,9 2,2 10,1 0,5 0,4 8,8 2,6 477* 5,9

174 2,5 1,3 1,9 0,6 0,2 18,9 1,0 4,0 0,6 970* 0,5 1,3 0,9 0,2 0,3 0,0 1,3 0,1 0,0 0,3 2,2 1,5 7,9 6,8 3,1 8,1 0,1 17,4 4,5

110 1,3 1,2 2,5 1,2 3,8 13,8 0,7 2,8 8,4 0,8 0,2 3,0 2,8 0,4 0,2 129* 0,7

0,2 8,4 0,0 0,6 0,5 0,3 0,3 0,3 1,4 0,6 0,0 0,1 0,0 0,8 0,6 1,9 0,6 0,5 0,4 64,7 1,8

0,4 71,9 2,2 1,5 2,6 0,9 0,4 38,9 2,0 6,7

18,0

5,7 5,2 1,9 3,3 1,7 0,8 29,0 0,9 31,2 16,8 19.1 1,8 2,1 3,4 11,2 25,3 25,1 10,1 106* 628*

72,3 0,3 0,6 0,3 0,0 0,0 0,7 0,0 0,1 12,2 0,5 2,0 2,7 2,3 0,7 2,5 16,0 0,8 0,2 2,4 27,1 2,9 1,1 0,8 0,2 0,5 15,5 0,0 0,6 0,4 0,6 2,7 1,6 2,0 0,3 0,4 47,4 0,0 0,8 0,2 25,0 1,8 1,5 3,9 0,8 0,4 5,3 1,3 40* 0,1 4535* 3,2 0,7 1,2 0,9 2,8 1598* 0,4 7,9 1172* 633*

1,3 2,6 1,6 0,5 0,4 3,4 0,5 0,1 0,0 3,1 1,9 1,6 1,5 0,5 0,8 17,9 0,1 0,1 21,1 8,0 2,2 1,1 2,3 1,2 2,1 7,8 0,7 0,3 0,1 225 0,6 0,5 0,3 0,3 0,3 1,4 0,6 0,0 0,1 0,0 1,2 1,5 1,1 0,4 0,6 0,0 23,3 12,1 29,8

9,2 1,7 1,1 1,4 0,9 0,7 1,9 1,0 0,0

1,4

1,4 0,7 0,9 0,8 1,4 2,3

51,5

0,8

34,4 11,0 0,6 3,4

1,5

2,3

1,5

11,6 730* 0,5

135* 212*

65,1

(2016)16:549 Page6of17

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