Expression of circadian clock genes and(3)

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）（续） B.在27个膀胱切除术患者中上调和下调的基因的mRNA基因表达的平均T / B倍数变化 c.非整倍体和二倍体患者肿瘤样品中mRNA基因表达的平均T / B倍数变化

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

B. Average T/B fold change in mRNA gene expression of genes upregulated and downregulated in 27 cystectomypatients Number of 17

17 4

3

7

11

8

13

13 11 20

19

14

16

19

19

20

8

8

22

8

13

15

19

patients

Average 2,47 1,67 1,70 1,50 3,34 1,99 1,72 37,63 1,57 4,08 2,56 20,69 5,44 2,60 2,65 1,69 2,91 3,32 6,69 129,69 13,30 67,51 143,45 382,79 up-regulation st.dev 1,2 0,6 0,2 0,2 2,4 0,8 0,7 61,5 0,6 3,4 1,0 37,2 9,9 1,9 1,8 0,6 2,2 3,7 8,2 361,9 22,1 129,9 328,1 1036,3 Number of 7

8 23

21

19

13

17

14

12 13 7

8

12

10

8

7

7

19

19

5

17

14

12

8 patients

Average 0,64 0,60 0,41 0,39 0,50 0,53 0,49 0,13 0,65 0,54 0,69 0,48 0,69 0,60 0,58 0,64 0,63 0,48 0,42 0,33 0,44 0,21 0,19 0,33 up-regulation st.dev

0,2

0,2

0,3

0,2

0,3

0,2

0,2

0,2

0,2

0,2

0,2

0,3

0,2

0,2

0,2

0,1

0,3

0,3

0,2

0,3

0,3

0,2

0,2

0,3

B2. Average T/B fold change in mRNA gene expression of genes upregulated and downregulated in 27 cystectomy patients. Patient samples identified as outliers by SPSS for respective gene assys have been excluded from the analysis (*)

B2。在27个膀胱切除术患者中上调和下调的基因的mRNA基因表达的平均T / B倍数变化。从分析中排除了通过SPSS鉴定为异常值的相应基因鉴定的患者样品（*） Number of 17

17 4

3

7

11

8

13

13 11 20

19

14

16

19

19

20

8

8

22

8

13

15

19

patients

Average 2,47 1,67 1,70 1,50 3,34 1,99 1,72 37,63 1,57 4,08 2,56 20,69 5,44 2,60 2,65 1,69 2,91 3,32 6,69 129,69 13,30 67,51 143,45 382,79 up-regulation st.dev 1,2 0,6 0,2 0,2 2,4 0,8 0,7 61,5 0,6 3,4 1,0 37,2 9,9 1,9 1,8 0,6 2,2 3,7 8,2 361,9 22,1 129,9 328,1 1036,3 Number of 7

8 23

21

19

13

17

14

12 13 7

8

12

10

8

7

7

19

19

5

17

14

12

8 patients

Average 0,64 0,60 0,41 0,39 0,50 0,53 0,49 0,13 0,65 0,54 0,69 0,48 0,69 0,60 0,58 0,64 0,63 0,48 0,42 0,33 0,44 0,21 0,19 0,33 up-regulation st.dev

0,2

0,2

0,3

0,2

0,3

0,2

0,2

0,2

0,2

0,2

0,2

0,3

0,2

0,2

0,2

0,1

0,3

0,3

0,2

0,3

0,3

0,2

0,2

0,3

C. Average T/B fold change in mRNA gene expression in aneuploid and diploid patient tumour samples Aneuploid(19patients)非整倍体 Average 1,9 1,3 0,6 0,6 1,2 1,2 0,8 12,3 1,2 2,1 2,2 13,7 2,0 2,1 2,4 1,4 2,3 1,3 2,9 137 5,7 43 111 325 St.dev 1,4 0,8 0,5 0,4 1,7 0,9 0,5 38,6 0,7 2,8 1,3 34,1 1,9 2,0 1,9 0,6 2,1 2,5 6,0 390 15,3 112 126 1032 Diploid (8 patients)

Average 1,6 1,3 0,6 0,6 1,4 1,2 1,2 32,1 1,0 1,9 1,7 17,0 6,0

1,1

1,2 1,3 2,3 1,3 0,9 31,1 0,9 7,9

5,4

137 st.dev

1,0

0,4

0,6

0,5

1,9

0,9

1,0

60,8

0,5

2,8

1,0

29,9 13,4 0,6

0,9

0,8

2,4

2,2

1,2

44,0

0,6

12,3 5,6

339

* Gene expression levels identified as outliers by SPSS statistical analysis

通过SPSS统计分析确定为异常值的基因表达水平

Litlekalsoyetal.BMCCancer(2016)16:549 Page7of17

Litlekalsoyetal.BMCCancer(2016)16:549 Page 8of 17

Fig. 1 (See legend on next page.)

Litlekalsoyetal.BMCCancer(2016)16:549 Page 9of 17

(See figure on previous page.) Fig. 1 Gene expression signal correlation plots. The plots display the correlations between mRNA normalized gene expression levels in the normal control bladder tissue samples of 15 patients with BPH (a), benign tissue peripheral to the tumour (b) and tumour tissue (c) of 27 cystectomypatients,respectively.Pearson’sproductmomentcorrelationcoefficients(r)foreachpairofsampleswerecalculatedusingDataAssist fromAppliedBiosystems.Eachcellrepresentsadifferentscatterplot,colouredtoindicatethestrengthofthecorrelationsbetweenthesamples. The higher the correlation between the gene expression levels in the two samples (the closer the correlation coefficient (r), is to 1), the colour moves towards brighter red. The poorer the correlation between the gene expression levels in the two samples (the closer r is to 0), the colour movestowardsdarkerredandthengreen,indicatingnocorrelation.Allsamplesarecorrelatedwitheachotherforeachoftheselectedgenes 基因表达信号相关图。该图显示了分别具有BPH（a），肿瘤良性组织（b）和肿瘤组织（c）的15名患者的正常对照膀胱组织样品中mRNA归一化的基因表达水平之间的相关性。使用来自Applied Biosystems的DataAssist计算每对样品的Pearson's乘积矩相关系数（r）。每个单元表示不同的散点图，其着色以指示样本之间的相关性的强度。两个样品中的基因表达水平之间的相关性越高（相关系数（r）越接近1），颜色朝向更亮的红色移动。两个样品中基因表达水平之间的相关性越差（r越接近0），颜色向更深的红色移动，然后向绿色移动，表明没有相关性。对于每个选择的基因，所有样品彼此相关

图2mRNA倍数变化基因表达图。相对于来自BPH患者的正常膀胱粘膜组织，良性相邻粘膜和肿瘤组织中的基因表达水平。相对量图显示取自膀胱切除术（27名患者）的良性（蓝色条）和肿瘤（红色条）组织与来自BPH患者的正常膀胱组织的mRNA水平的log2倍变化。在一个。显示时钟基因的mRNA水平的log2倍数变化（log2 RQ），而肿瘤标记基因在b中绘制。具有负值的基因被下调，而具有正值的基因在恶性膀胱（肿瘤和良性组织）中相对于正常膀胱（其每个基因的log2值为0）上调。对于KRT7，PER1，PER2，PTEN，uPAR和PAI-1，发现p值≤0.05的统计学显着性（两个样本，双尾学生t检验）

Fig. 2 mRNA fold change gene expression plots. Gene expression levels in benign neighbouring mucosa and tumour tissue relative to normal bladder mucosa tissue from BPH patients. The relative quantity plots display the log2 fold change in mRNA levels in the benign (blue bars) and tumour(redbars)tissuetakenfromcystectomies(27patients)versusnormalbladdertissuefromBPHpatients.Thebarsina.displaythelog2fold change (log2 RQ) in mRNA levels of the clock genes, while the tumour marker genes are plotted in b. Genes with a negative value are down- regulated, while genes with a positive value are up-regulated in the malignant bladder (tumour and benign tissue) versus the normal bladder (whoselog2valueis0foreachgene).Statisticalsignificancewithap-value≤0.05wasfoundforKRT7,PER1,PER2,PTEN,uPARandPAI-1(Two-sam- ple, two-tailed Student’st-test)

Litlekalsoyetal.BMCCancer(2016)16:549 Page 10 of 17

Fig. 3 Relative mRNA quantity of PER1, PER2, PER3 and CRY2. Real-time quantitative PCR expression levels normalized against the endogenous control β-actin (ACTB). The figure gives the comparison between 27 tumour and matched benign bladder tissue samples. Columns, median; bars, a: PER1, b: PER2, c: PER3 and d: CRY2. The relative gene expression of all four genes was significantly elevated in the benign versus malignant bladder tissue. The changes were consistent for each pair of tumour - neighbouring mucosa, indicated by the p-value of the statistical test (non- parametric paired samples Mann-Whitney test)

图3PER1，PER2，PER3和CRY2的相对mRNA量。相对于内源对照β-肌动蛋白（ACTB）标准化的实时定量PCR表达水平。该图给出了27个肿瘤和匹配的良性膀胱组织样品之间的比较。柱，中值; 条，a：PER1，b：PER2，c：PER3和d：CRY2。所有四个基因的相对基因表达在良性与恶性膀胱组织中显着升高。对于每对肿瘤 - 相邻粘膜的变化是一致的，由统计检验的p值（非参数配对样本Mann-Whitney检验）Gene expression correlation plots基因表达相关图

from cystectomies compared with normal bladder mucosa The strength of the correlations of relative mRNA-levels in the different patient samples is visualized in the gene expression signal correlation plots (Fig. 1). The plots display the strength of the correlations betweennormalisedgeneexpressionlevelsin15biopsiesofnormal bladder mucosa (Fig. 1a), and 27 matched benign/tumour biopsies taken from patients who underwent cystectomy (Fig.1bandc,respectively).Anincreasingdissimilarityin gene expression levels and poorer correlations among patients were seen when moving from normal bladder mucosa to neighbouring and tumour tissue. 不同患者样品中相对mRNA水平的相关性的强度在基因表达信号相关图中可视化（图1）。这些图显示了正常膀胱粘膜（图1a）活组织检查的15个标准化基因表达水平与从膀胱切除术（图1b和c）获得的27个匹配的良性/肿瘤活组织检查的相关性的强度。当从正常膀胱粘膜移动到相邻和肿瘤组织时，观察到患者中基因表达水平的增加的不相似性和较差的相关性。

Gene expression patterns (Ct-values) of the normal un- related mucosa (15 samples) were consistent regarding the two housekeeping genes included in the study. The gene expressions in the tumour and neighbouring tissue collected from the cystectomies were, for all genes included, compared relatively to the gene expression pattern of these 15 samples. 来自囊肿切除术的肿瘤/相邻粘膜中的mRNA基因表达与正常膀胱粘膜相比正常不相关粘膜（15个样品）的基因表达模式（Ct值）对于包括在研究中的两个管家基因是一致的。对于所有包括的基因，从囊性切除术收集的肿瘤和相邻组织中的基因表达相对于这15个样品的基因表达模式进行比较。

BMAL1 was down-regulated in both neighbouring and tumour tissue compared to normal mucosa, while CLOCKwas slightly up-regulated in neighbouring tissue and slightly down-regulated in tumour (Fig. 2). 与正常粘膜相比，BMAL1在相邻和肿瘤组织中下调，而CLOCK在邻近组织中轻微上调并在肿瘤中轻微下调（图2）。

mRNA gene expressions in tumour/neighbouring mucosa

Litlekalsoyetal.BMCCancer(2016)16:549 Page 10 of 17

Fig. 4KRT7，KRT14，NRAS，TP53和UPAR的相对mRNA量。相对于内源对照β-肌动蛋白标准化的实时定量PCR表达水平。该图给出了27个肿瘤和匹配的良性膀胱组织样品之间的比较。柱，中值; bar，a：KRT7，b：KRT14，c：NRAS，d：TP53和e：UPAR。细胞角蛋白，NRAS和TP53的基因表达水平在肿瘤与良性膀胱组织中显着升高，而与肿瘤相比，良性组织中UPAR的表达显着升高。对于每对肿瘤 - 相邻粘膜的变化是一致的，由统计检验的p值（非参数配对样本Mann-Whitney检验）

Fig.4RelativemRNAquantityofKRT7,KRT14,NRAS,TP53andUPAR.Real-timequantitativePCRexpressionlevelsnormalizedagainsttheendogenous controlβ-actin.Thefiguregivesthecomparisonbetween27tumourandmatchedbenignbladdertissuesamples.Columns,median;bars,a:KRT7, b:KRT14,c:NRAS,d:TP53ande:UPAR.Thegeneexpressionlevelsofthecytokeratins,theNRASandTP53weresignificantlyelevatedinthetumourversus benignbladdertissue,whiletheexpressionofUPARwassignificantlyelevatedinthebenigntissuecomparedtothetumour.Thechangeswereconsistent foreachpairoftumour-neighbouringmucosa,indicatedbythep-valueofthestatisticaltest(non-parametricpairedsamplesMann-Whitneytest)

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