Expression of circadian clock genes and(4)

Litlekalsoyetal.BMCCancer(2016)16:549 Page 11of 17

Fig. 5 Unsupervised hierarchical cluster analysis of differentially expressed genes. Normal bladder tissue from 15 unrelated donors with BPH together with tumour and matched benign tissue from 27 cystectomy patients were analysed. Real-time qPCR expression data were imported into DataAssist (ABI) for unsupervised hierarchical cluster analysis. Distances between samples and assays were calculated based on the delta-Ct values using Pearson’s correlation. Differentially expressed genes are represented in rows and the different samples are represented in columns. Each cell in the heat map represents one samples relative expression of one gene. For each gene assay, the middle expression level was set as the median of all of the delta-Ct-values of all samples for that gene assay. Gene expression colour codes in the heat map: Green colour represents relative levels of mRNA lower than the middle value for that gene expression assay (decreased gene expression); Red colour represents levels of mRNA higher than the middle expression level (increased gene expression); Dark colour reflects an mRNA expression level closer to the middle expression level (no major increase or decrease in gene expression). Patient samples: Blue colour: The normal bladder tissue taken from BHP patients is numbered 1–15N; Green colour: Normal benign tissue taken peripherally to the tumour is numbered 1–27N; Red colour: tumour tissue is numbered 1–27T. Genes: Purple colour: Clock genes; Black colour: cancer associated genes. (Clustering method: complete linkage. Map type: assay centric)

图5 差异表达基因的无监督层次聚类分析。分析来自15个不相关供体的BPH的正常膀胱组织以及来自27个膀胱切除患者的肿瘤和匹配的良性组织。将实时qPCR表达数据导入用于无监督层次聚类分析的

DataAssist（ABI）。使用Pearson相关性基于Δ-Ct值计算样品和测定之间的距离。差异表达的基因以行表示，不同的样品以列表示。热图中的每个细胞代表一个基因的一个样品相对表达。对于每个基因测定，将中间表达水平设定为该基因测定的所有样品的所有Δ-Ct值的中值。热图中的基因表达颜色代码：绿色代表低于该基因表达测定的中间值的mRNA的相对水平（降低的基因表达）; 红色代表高于中间表达水平的mRNA水平（增加的基因表达）; 深色反映更接近中间表达水平的mRNA表达水平（基因表达没有大的增加或减少）。患者样品：蓝色：取自BHP患者的正常膀胱组织编号为1-15N; 绿色：在肿瘤周围取得的正常良性组织编号为1-27N; 红色：肿瘤组织编号为1-27T。基因：紫色：时钟基因; 黑色：癌症相关基因。（聚类方法：完全连锁。地图类型：测定中心）

PER1 and CRY1 were both up-regulated in neighbour- ing and tumour tissue compared to normal mucosa,while PER2 and PER3 were up-regulated in neighbouring mucosa and down-regulated in the tumour tissue. CRY2 was down-regulated in both tissue types compared with normal mucosa. This corresponds well with the immu- nostaining results (Table 3). 与正常粘膜相比，PER1和CRY1在相邻和肿瘤组织中都上调，而PER2和PER3在相邻粘膜中上调，在肿瘤组织中下调。 CRY2在两种组织类型相比正常粘膜下调。这与免疫结果良好（表3）。

The casein kinases CSNK1A1L and CSNK1E weredown- regulated in neighbouring mucosa and up-regulated in tumour tissue, while CSNK1A1 was down-regulated inboth tissue types (Fig.2a). 酪蛋白激酶CSNK1A1L和CSNK1E在相邻粘膜中下调，在肿瘤组织中上调，而CSNK1A1在两种组织类型中下调（图2a）。

HRAS was down-regulated in neighbouring and tumour tissue compared to the normal mucosa, while NRAS seemed to be equally down-regulated in neighbouring and up-regulated in tumour tissue. KRAS, EGFR and p16were all up-regulated in both tissue types compared to normal mucosa (Fig. 2b). The tumour suppressors TP53 and PTEN were moderately down-regulated in both tissue types and uPAR and PAI-1 displayed similar patterns. Cytokeratin 1 (KRT1) was down-regulated in neighbour- ing and up-regulated in tumour tissue, while the other cytokeratins (KRT5-7-10-14) were all up-regulated in both tissue types compared with normal mucosa. Only KRT7, PER1,PER2,uPAR,PTENandPAI-1hadp-valuesbelow 0.05. This might be explained by the heterogeneity of the patient samples individual gene expression levels, but the tendency described between the biological groups seemed clear. 与正常粘膜相比，HRAS在邻近和肿瘤组织中下调，而NRAS似乎在肿瘤组织中在相邻和上调中同样下调。与正常粘膜相比，KRAS，EGFR和p16在两种组织类型中都上调（图2b）。肿瘤抑制剂TP53和PTEN在两种组织类型中适度下调，并且uPAR和PAI-1显示相似的模式。细胞角蛋白1（KRT1）在肿瘤组织中被下调和上调，而其他细胞角蛋白（KRT5-7-10-14）与正常粘膜相

Litlekalsoy比在两种组织类型中都被上调。只有etal.BMCCancer(2016)16:549

KRT7，PER1，PER2，uPAR，PTEN和PAI-1具有低于0.05的p值。这可以通过患者样品的个体基因表达水平的异质性来解释，但是在生物学组之间描述的趋势似乎是清楚的。

Differences in mRNA gene expression levels in tumour versus benign neighbouring mucosa from cystectomies 肿瘤中mRNA基因表达水平与来自膀胱切除术的良性相邻粘膜的差异 Average down- or up-regulation with standard deviation (SD) of each gene expression studied is given in Table 5B. Theclockandclockrelatedgenes(BMAL,CLOCK,PER1, PER2, PER3, CRY1, CRY2, CSNK1A1, CSNK1E) tendedto be either up-regulated or down-regulated from 2-fold to 5-fold in tumour samples compared with matched benign samples. CSNK1A1L, which is a homolog to CSNK1A1, showed a much higher fold change, from approximately thirty-fold to more than one hundred fold up-regulation in 6 out of 27 patient samples, as well as being not de- tected and highly down-regulated in a subset of patients.In the majority of the samples, the expression of BMAL and CLOCK was down-regulated in the tumour tissue compared to matched benign mucosa. PER1, PER2 and PER3 were lower in the tumour when compared to neigh- bouring benign mucosa. For CRY1, the gene seemed to be equally up- or down-regulated in the samples, and for CRY2, the majority of the samples showed a down- regulation in the tumour tissue. For the three clockrelatedcasein kinases, the samples were almost equally distrib- uted between up- and down-regulated gene expression inthe tumour tissue, with a wide variation in gene expression evels and hence T/B-ratios. 具有所研究的每种基因表达的标准偏差（SD）的平均下调或上调在表5B中给出。肿瘤样本中的时钟和时钟相关基因（BMAL，CLOCK，PER1，PER2，PER3，CRY1，CRY2，CSNK1A1，CSNK1E）倾向于上调或下调，从2倍到5倍良性样品。作为CSNK1A1的同源物的CSNK1A1L显示出高得多的倍数变化，在27个患者样品中的6个中从大约三十倍上调至超过一百倍，以及未检测到和高度下调，在一部分患者中被调节。在大多数样品中，BMAL和CLOCK的表达在肿瘤组织中与匹配的良性粘膜相比下调。与邻近的良性粘膜相比，PER1，PER2和PER3在肿瘤中较低。对于CRY1，该基因似乎在样品中同样上调或下调，对于CRY2，大多数样品在肿瘤组织中显示下调。对于三种时钟相关的酪蛋白激酶，样品在肿瘤组织中上调和下调基因表达之间几乎平均分布，基因表达水平具有广泛的变化，因此T / B比率。

The mRNA levels for the common tumour markers showed that p16 was moderately to highly up-regulated in19 of the 27 samples, while PTEN was mainly moderatelyup-regulated or down-regulated in half the samples eachTP53, EGFR, NRAS, HRAS, KRAS, UPAR and PAI-1 wasgenerally approximately 2-fold down-regulated or between2- and 6-fold up-regulated, with some extreme exceptionsThe cytokeratins were different from the other genes stud-ied, displaying extremely high T/B-fold changes (100-to1000-fold up-regulated or highly down-regulated) in subsets of tumours. KRT1 was mainly down-regulated (17/27of the samples), while KRT7 and KRT14 were

mainly up-regulated. KRT5 and KRT10 were up- and down-Page 12of 17

regulatedin approximately half of the samples, respectively. 普通肿瘤标志物的mRNA水平显示，在27个样品中的19个中，p16中等至高度上调，而PTEN在一半样品中主要是中等上调或下调，每个TP53，EGFR，NRAS，HRAS， KRAS，UPAR和PAI-1通常约2倍下调或2和6倍上调，有一些极端的例外细胞角蛋白与所研究的其他基因不同，显示极高的T / B 在肿瘤亚组中的倍数变化（100至1000倍上调或高度下调）。 KRT1主要下调（17/27样品），而KRT7和KRT14主要上调。 KRT5和KRT10分别在约一半的样品中上调和下调

Among the clock genes, the expression of PER1, PER2,PER3 and CRY2 were significantly elevated in the benigntissue compared to the tumour tissue (p = 0.001, 0.002,0.037 and 0.001 respectively) (Fig. 3). The relative quantityof mRNA was significantly elevated in the tumour tissuecompared to the benign tissue for KRT7, KRT14, NRASand TP53 (p = 0.004, 0.010, 0.008 and 0.004, respectively).This also corresponds with Fig. 2b which reveals the samepattern. The expression of TP53 is lower in the neighbour-ing mucosa compared to tumour tissue and even more down-regulated in the normal unrelated mucosa. For uPAR, the level of mRNA was statistically elevated in the benign tissue compared to the tumour (p = 0.019) (Fig. 4), this is also in accordance with the expressions pattern dis- played in Fig. 2b.

在时钟基因中，与肿瘤组织相比，良性组织中PER1，PER2，PER3和CRY2的表达显着升高（分别为p = 0.001,0.002,0.037和0.001）（图3）。与KRT7，KRT14，NRAS和TP53的良性组织相比，肿瘤组织中mRNA的相对量显着升高（分别为p = 0.004,0.010,0.008和0.004）。这也对应于图2。 2b，其揭示相同的图案。与肿瘤组织相比，TP53的表达在相邻粘膜中较低，在正常无关的粘膜中甚至更下调。对于uPAR，与肿瘤相比，良性组织中mRNA的水平统计学上升高（p = 0.019）（图4），这也与图2b中显示的表达模式一致。

Litlekalsoyetal.BMCCancer(2016)16:549 Page 13of 17

Table 6 Correlations between the different clock genes

.

.

-

Statistical correlations统计相关

Spearman’s rank correlation revealed correlations of the estimated T/B ratios between the various clock-genes. The ones found statistically significant, are listed in Table 6. Statistical significance between the tumour associated genes is listed in Table 7, and correlations between the clock genes compared to other cancer-associated genes are listed in Table 8. The relative quantity of mRNA in tumour compared to neighbouring mucosa was found statistically significant for the genes displayed in Figs. 3 and 4. 斯皮尔曼秩相关揭示了各种时钟基因之间的估计T / B比率的相关性。发现具有统计学显着性的那些，列于表6中。肿瘤相关基因之间的统计学显着性列于表7中，并且时钟基因与其他癌症相关基因相比的相关性列于表8中。肿瘤与邻近的粘膜相比在图3和图4中显示的基因具有统计学显着性。

Hierarchical cluster analysis分层聚类分析

An unsupervised hierarchical cluster analysis of the relative mRNA-levels was performed and visualized in a heat map (Fig. 5). There were substantial variations between normal mucosal and tumour expression pat- terns. The neighbouring mucosa exhibited a series of aberrations similar to the tumour and appeared consid- erably different from the unrelated donor mucosa. 进行相对mRNA水平的无监督层次聚类分析，并在热图中显现（图5）。正常粘膜和肿瘤表达模式之间存在显着差异。相邻的粘膜表现出一系列类似于肿瘤的畸变，并且显着地不同于不相关的供体粘膜。

Genesencoding

p-value

Correlation coefficient, C

Stimulatory

BMAL1

- CLOCK 0.004 0.539 - CSNK1A1 0.003 0.544 - CSNK1E

0.002 0.566 CLOCK

- PER3 0.001 0.593 - CRY1 0.005 0.522 - CRY2 0.014 0.467 - CSNK1A1 0.029 0.421 - CSNK1E

0.013

0.471

Inhibitory

PER1 - CRY2 0.007 0.509 - CSNK1A1L 0.049 ?0.382 PER2 - CSNK1A1 0.001 0.620 - CSNK1E 0.009 0.495 PER3 - CRY1 0.012 0.475 - CRY2 0.000 0.687 CRY1 - CRY2 0.000 0.643 - CSNK1E 0.014 0.469 Casein kinases

CSNK1A1 - CSNK1E

0.000

0.900

The correlation coefficients: C < 0.3: poor correlation, 0.3 < C < 0.5: fair correlation, 0.6

0.6

The genes uPAR and PAI-1 clustered and connected to a cluster of p16 and KRT7. Five of the clock geneswere also clustered (PER1, PER2, PER 3, CRY1 and CRY2). CLOCK clustered with H-K-N-RAS, EGFR and TP53. They clustered with the two cytokeratins (KRT5 and KRT10), which in turn were connected to KRT14.The casein kinases CSNK1A1 and CSNK1E clustered and connected to the cluster of BMAL1 and PTEN, where- upon these clusters were connected to the cluster of CSNK1A1L andKRT1.

基因uPAR和PAI-1聚集并连接到p16和KRT7的簇。五个时钟基因也聚集（PER1，PER2，PER 3，CRY1和CRY2）。 CLOCK与H-K-N-RAS，EGFR和TP53聚类。它们与两种细胞角蛋白（KRT5和KRT10）聚集，其依次与KRT14连接。酪蛋白激酶CSNK1A1和CSNK1E聚集并连接到BMAL1和PTEN的群集，其中这些群集连接到CSNK1A1L和KRT1的群集。

Sorted by tissue type, all the normal bladder samples, except for one (11N blue), clustered together. This out-lier was placed among the neighbouring samples. There was a similar expression pattern between 6N blue, the outlier, and its adjacent tumour sample (23T red). They seemed to have a lower level of mRNA expression for all genes selected, and all samples in this cluster revealed a low expression of uPAR and PAI-1 (which were strongly correlated;p=0.00,c=0.781).

Litlekalsoy按组织类型分类，所有正常膀胱样品，除了一个etal.BMCCancer(2016)16:549

（11N蓝色），聚集在一起。这个样品被放置在相邻的样品之间。在6N蓝色，异常值和其相邻肿瘤样品（23T红色）之间存在相似的表达模式。对于所有选择的基因，它们似乎具有较低水平的mRNA表达，并且该簇中的所有样品显示uPAR和PAI-1的低表达（其是强相关的; p = 0.00，c = 0.781）。

The neighbouring samples from the cystectomies were mainly divided into two clusters. In the first, 12 of the neighbouring samples clustered with four tumour sam- ples (5, 7, 9 and 13T). This cluster revealed a lower ex- pression or minor changes in the expression ofBMAL1,CLOCK, tumour marker genes, cytokeratins and casein kinases. 来自囊肿切除术的相邻样品主要分为两个簇。在第一个，12个相邻的样本聚集与四个肿瘤样本（5,7,9和13T）。这个聚类显示BMAL1，CLOCK，肿瘤标志物基因，细胞角蛋白和酪蛋白激酶的表达较低的表达或较小的变化。 The majority of these samples had a higher ex- pression of PER1, PER2, PER3, CRY1 and CRY2. In the second cluster (8 neighbouring samples, 23T and 11N blue), CLOCK, HRAS, KRAS, NRAS, TP53, EGFR andcytokeratin 14, revealed a lower level of expression/ minor changes in gene expression. 这些样品中的大多数具有更高的PER1，PER2，PER3，CRY1和CRY2的表达。在第二簇（8个相邻样品，23T和11N蓝色），CLOCK，HRAS，KRAS，NRAS，TP53，EGFR和细胞角蛋白14中，揭示了较低水平的表达/基因表达的微小变化。 Except for 23T and 11N blue, the neighbouring samples in this cluster also revealed a higher expression of uPAR, PAI-1, p16,KRT7, PER1,PER2,PER3,CRY1andCRY2.Mostofthetumour samples accumulated into one cluster (17 samples). One neighbouring sample (14N green) was included in this sub-group. Lower expression of CLOCK, the stimulatory clock genes and PTEN, together with increased expres- sion of KRT7 and KRT14, characterized this cluster.Some aneuploid tumours (15, 17, 19, 21, 22, and 27T) grouped together in a sub-cluster, with increased expres- sion of HRAS, KRAS, NRAS, TP53 and EGFR. A mixed cluster of tumour and neighbouring mucosal samples (normalgreen:9,10,23,24;tumourred:3,10,12,24)

revealed higher expression of tumour markers, cytokera- Page 14of 17

tins and casein kinases. 除了23T和11N蓝色，该簇中的相邻样品还显示uPAR，PAI-1，p16，KRT7，PER1，PER2，PER3，CRY1和CRY2的更高表达。大多数肿瘤样品累积为一个簇（17个样品）。在该亚组中包括一个相邻样品（14N绿色）。低表达的时钟，刺激时钟基因和PTEN，加上表达的KRT7和KRT14，表征这个群集。一些非整倍体肿瘤（15,17,19,21,22和27T）组合在一个亚簇中，增加了HRAS，KRAS，NRAS，TP53和EGFR的表达。肿瘤和相邻粘膜样品的混合簇（正常绿色：9,10,23,24;肿瘤红：3,10,12,24）显示肿瘤标志物，细胞因子和酪蛋白激酶的更高表达。

Litlekalsoyetal.BMCCancer(2016)16:549 Page 15of 17

Table 7 Correlations between the selected tumour markers表7所选肿瘤标记物之间的相关性

Genes encoding TP53

-PTEN - HRAS - KRAS - NRAS - UPAR - PAI-1 - KRT7 - KRT14

p-value 0.042 0.005 0.000 0.000 0.000 0.011 0.017 0.011

Correlation coeff, C 0.395 0.522 0.654 0.660 0.627 0.482 0.455 0.485

Genesencoding HRAS

-NRAS -UPAR -PAI-1 -KRT7 -KRT5 -KRT14 -NRAS - UPAR -KRT7 -KRT5 -KRT14 -KRT7 -KRT5 -KRT14 - PAI-1 -KRT7 -KRT5 -KRT14

p-value 0.011 0.026 0.017 0.004 0.002 0.000 0.000 0.000 0.041 0.001 0.002 0.035 0.006 0.011 0.000 0.005 0.001 0.006

Correlation coeff, C 0.479 0.428 0.455 0.536 0.569 0.637 0.701 0.627 0.396 0.605 0.561 0.407 0.518 0.479 0.781 0.525 0.599 0.519

KRAS NRAS

P16 -KRAS - NRAS - KRT14

PTEN -KRAS - NRAS - UPAR - PAI-1 - KRT7 - KRT5 - KRT10

EGFR -HRAS - NRAS - KRT14

0.024 0.001 0.028 0.022 0.047 0.008 0.027 0.002 0.025 0.006 0.005 0.013 0.006

0.432 0.603 0.424 0.438 0.386 0.503 0.425 0.570 0.430 0.511 0.526 0.471 0.511

PAI-1

UPAR

KRT7

-KRT5 -KRT10 -KRT10 -KRT14

0.017 0.017 0.002 0.002

0.456 0.457 0.562 0.576

KRT5

The correlation coefficients: C < 0.3: poor correlation, 0.3 < C < 0.5: fair correlation, 0.6 < C < 0.8: moderately strong correlation and 0.8 < C: Very strong correlation

Correlations between gene expressions and DNA ploidy 基因表达与DNA倍体之间的相关性 Histological stage and vascular invasion are listed in Table 1. Diploid/aneuploid DNA stemline values are shown in Tables 5C and 9. According to the ploidy of the cancer cells, the average tumour/benign fold change in mRNA levels were similarly expressed for the clock genes exceptforBMAL1,CRY2andCSNK1A1L.Theaverageexpression of BMAL1 was slightly up-regulated in the aneuploid cells while CRY2 was slightly down-regulated for the aneuploid cells and up-regulated in the diploid cells. The average for CSNK1A1L was up-regulated for both categories, but more than the double for the diploidcancer cells (Table5C). 组织学分期和血管侵袭列于表1中。二倍体/非整倍体DNA干酪素值示于表5C和9.根据癌细胞的倍数，mRNA水平的平均肿瘤/良性倍数变化类似地表示为时钟基因除了BMAL1，CRY2和CSNK1A1L。 BMAL1的平均表达在非整倍体细胞中轻微上调，而CRY2对于非整倍体细胞轻微下调并在二倍体细胞中上调。CSNK1A1L的平均值上调两种类型，但大于二倍体癌细胞的双倍（表5C）。

For the other cancer related genes, the total T/B aver- ages for p16 and PTEN were found divergent in the two categories; with four fold higher expression in the dip-loid compared to the aneuploid stem line. The oppositepattern was seen for EGFR andHRAS, with an averageof two fold higher expression in the aneuploid comparedto the diploid cells. The average of the PAI-1 was slightly down-regulated in the diploid category and almost tree fold up-regulated in the aneuploid cells. Due to individ- ual samples with very high T/B ratios, it was difficult to estimate the cytokeratins’ average in tumour/benign tis- sue. However, the trend among the five cytokeratins re- vealed an increased (several T/B-fold) level of gene expression in the aneuploid as compared to the diploid cancercells. 对于其他癌症相关基因，p16和PTEN的总T / B平均值在两个类别中发现不同; 与非整倍体干细胞系相比，在浸染液中表达高四倍。对于EGFR和HRAS观察到相反的模式，与二倍体细胞相比，在非整倍体中平均表达高两倍。 PAI-1的平均值在二倍体类别中轻微下调，并且在非整倍体细胞中几乎树状上调。由于个体样品具有非常高的T / B比，很难估计肿瘤/良性组织中细胞角蛋白的平均值。然而，五种细胞角蛋白的趋势表明与二倍体癌细胞相比，非整倍体中基因表达水平增加（几T / B倍）

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