Type of Manuscript: Researcharticle

Eissa et al.,2015

Type of manuscript: ResearchArticle

Integrative Functional Genetic-Epigenetic Approach for Selecting Genes as urine Biomarkers for Bladder Cancer Diagnosis.

Running title : Detection of Urinary Panel of Biomarkers in Bladder Cancer

SanaaEissa1 ,Marwa Matboli1,Nada O.E. Essawy2,Youssef M.Kotb3

1 Oncology Diagnostic Unit, Medical Biochemistry and Molecular biology Department, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt, P.O. box 11381.

2MSc. Pharmacogenetics and Stratified Medicine, University College London.

3Urology Department, Faculty of Medicine, Ain Shams University,Cairo, Egypt.

#Corresponding author:

Sanaa Eissa1, 1Oncology Diagnostic Unit, Medical Biochemistry and Molecular biology Department, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt, P.O. box 11381..

2. Patients and methods

2.7. qRT-PCR analysis of miRNA-96 and miRNA210 expression

The PCR program was as follow: firstly, denaturation at 95°C for 15 min; followed by 40 cycles of denaturation for 10 sec at 94°C; then annealing for 30 sec at 55°C; lastly, extension for 34 sec at 70°C. Each reaction was carried out in triplicate.The threshold cycle (Ct) value of each sample was calculated usingStepOnePlus™ software v2.2.2 (Applied Biosystems). Amplification plots and Tm values were analyzed to confirm the specificities of the amplicons for PCR array amplification.The results were analyzed by the melting curve analysis software of Applied Biosystem. Amplification plots and Tm values were analyzed to confirm the specificities of the amplicons for SybrGreen -based PCR amplification.

2.9. Statistical analysis

Comparisons were performed using Chi squared and T-tests or ANOVA tests, as appropriate. Associations of the selected epigenetic biomarkers andHYAL1 protein with clinicopathologic variables were analyzed using non parameteric Mann–Whitney U test or Kruskal Wallis test. The cut-off scores for the analysis were obtained by analyzing the ROC curve to determine whether there was a significant difference for the chosen biomarkers between the investigated groups.Pearson's correlation analysis was performed to determine whether there were correlations between the urinary levels of biomarkers in bladder cancer versus control. P value< 0.01 was considered statistically significant.

RESULTS

Supplementary tables:

Supplementary Table 1: Relation between investigated urine markers and clinicopathological factors of bladder cancer patients

Groups
(n=94) / miR-210 / MiR-96 / LncRNA-UCA1 / HYAL1 protein / Positive cytologyb
(n=48)
qPCR / Cases>cutoff
(n=72) / qPCR / Cases>cutoff / qPCR / Cases>cutoff / Semiquantitaivezymography / Cases>cutoff
Mean Rank(RQ values)a / No (%)b / Mean Rank(RQ values)a / Positive b
(n=72) / Mean Rank(RQ values)a / Positive b
(n=86) / Mean Rank)a / Positiveb
(n=84)
Age / 42,17 / 32 (44.4%) / 39,42 / 30
41.7% / 46,42 / 42
48.8% / 45,21 / 42
50% / 30
62.5%
>61 years (n=48)
<61years(n=46) / 53.07 / 40
56.6% / 55.93 / 42
58.3% / 48.63 / 44
51.2% / 49.98 / 42
50% / 18
37.5%
p / 0.053 / 0.02* / 0.003** / 0.001* / 0.694 / 0.157 / 0.405 / 0.55 / 0.023*
Sex / 46.3 / 48
66.7% / 47.37 / 44
61.1% / 47.24 / 58
67.4% / 45.85 / 54
64.3% / 36
75%
Male (n=62)
Female (n=32) / 49.8 / 24
33.3% / 47.75 / 28
38.9% / 48 / 28
32.6% / 50 / 30
35.7% / 12
25%
p / 0.555 / 0.793 / 0.949 / 0.073 / 0.898 / 0.319 / 0.415 / 0.322 / 0.059
Smoking / 45,75 / 34
47.2% / 44,92 / 36
50% / 46,88 / 44
51.2% / 52,75 / 44
52.4% / 24
50%
Positive (n=48)
Negative (n=46) / 49.33 / 38
52.8% / 50.2 / 36
50% / 48.15 / 42
48.8% / 42.02 / 40
47.6% / 24
50%
p / 0.525 / 0.178 / 0.348 / 0.709 / 0.820 / 0.95 / 0.056 / 0.459 / 0.833
Bilharziasis / 43,92 / 30
41.7% / 38,92 / 26
36.1% / 54,66 / 38
44.2% / 43,39 / 32
38.1% / 20
41.7%
Positive (n=38)
Negative (n=56) / 49.93 / 42
58.3% / 53.32 / 46
63.9% / 42.64 / 48
55.8% / 50.29 / 52
61.9 / 28
58.3%
p / 0.294 / 0.657 / 0.012* / 0.123 / 0.036* / 0.015* / 0.229 / 0.182 / 0.802
Pathological type / 46,99 / 56
77.8% / 48,09 / 56
77.8% / 49,01 / 70
81.4% / 45,99 / 66
78.6% / 30
62.5%
TCC(n=74)
SCC(n=20) / 49.4 / 16
22.2% / 45.3 / 16
22.2% / 41.9 / 16
18.6% / 53.1 / 18
21.4% / 18
37.5%
p / 0.725 / 0.685 / 0.684 / 0.685 / 0.3 / 0.038* / 0.3 / 0.917 / 0.001**
Clinical Stage / 48,07 / 36
50% / 53,76 / 38
52.8 % / 46,89 / 44
51.2% / 45,89 / 40
47.6% / 18
37.5%
Earlystage (0+1)(n=46)
Latestage (II+111)(n=48) / 46.96 / 36
50% / 41.5 / 34
47.2% / 44.08 / 42
48.8% / 49.04 / 44
52.4% / 30
62.5%
p / 0.844 / 0.709 / 0.029* / 0.178 / 0.832 / 0.157 / 0.575 / 0.459 / 0.023*
Histological Grade / 45,63 / 12
16.7% / 54.13 / 14
19.4% / 48,13 / 16
18.6% / 37 / 14
16.7% / 6
12.5%
1(n=16)
2(n=52) / 48.35 / 40
55.6% / 44,77 / 38
52.8% / 46.81 / 48
55.8% / 49,69 / 48
57.1% / 26
54.2%
3(n=26) / 46,96 / 20
27.8% / 44.88 / 20
27.8% / 48,50 / 22
25.6% / 49.58 / 22
26.2% / 16
33.3%
p / 0.934 / 0.986 / 0.456 / 0.491 / 0.962 / 0.211 / 0.239 / 0.563 / 0.310

*Significant difference was detected between investigated groups at P<0.001 using Chi-square testa and non-parametric testb.TCC: Transitional cell carcinoma; SCC: Squamous cell carcinoma.

Urine marker / Cytology
Positive(n=50) / Negative(n=160)
MiR-210
Positive(n=80)
Negative(n=130) / 36
14 / 44
116
2 =31.989, P :0.001**
Urine marker / Cytology
Positive(n=50) / Negative(n=160)
MiR-96
Positive(n=84)
Negative(n=126) / 36
14 / 48
112
2 =28, P :0.001**
Urine marker / Cytology
LncRNA-UCA1
Positive(n=90)
Negative(n=120) / Positive(n=50) / Negative(n=160)
44
6 / 46
114
2 =54.6, P <0.001**
Urine marker / Cytology
HYAL1 protein / Positive(n=50) / Negative(n=160)
Positive(n=94)
Negative(n=116) / 44
6 / 50
110
2 =49.62, P <0.001**

Supplementary Table 2s: Concordance between investigated urine markers and cytology among all investigated groups (n = 210)

**Highly Significant difference was detected between investigated groups at P<0.001 using Chi-square test.miR: microRNA, lncRNA-UCA1: long non-coding RNA-Urothelial Cancer Associated 1, HYAL1: hyaluronoglucosaminidase 1.

Urine marker / HYAL1
Positive(n=94) / Negative(n=116)
MiR-210
Positive(n=80)
Negative(n=130) / 74
20 / 6
110
2 =119.11, P :<0.001**
Urine marker / HYL1
Positive(n=84) / Negative(n=10)
MiR-96
Positive(n=84)
Negative(n=126) / 82
12 / 2
114
2 =158.19, P :<0.001**
Urine marker / HYAL1
LncRNA-UCA1
Positive(n=90)
Negative(n=120) / Positive(n=84) / Negative(n=10)
80
14 / 10
106
2 =124.03, P <0.001**

Table 3s: Concordance between HYAL1 and Other urinary biomarkers among all investigated groups (n = 210)

**Highly Significant difference was detected between investigated groups at P<0.001 using Chi-square test.miR: microRNA, lncRNA-UCA1: long non-coding RNA-UrothelialCancer Associated 1, HYAL1: hyaluronoglucosaminidase 1.

Supplementary Table 4s: Concordance between miR-210, miR-96 and otherinvestigated urine markers among all groups of study (n = 210)

Urine marker / MiRNA-210 / MiRNA-96
Positive
(n=80) / Negative
(n=130) / Positive
(n=84) / Negative
(n=126)
LncRNA-UCA1
Positive(n=90)
Negative(n=120) / 74
6 / 16
114 / 70
14 / 20
106
2 =130.045, P :0.001** / 2 =93.657, P :0.001**
Urine marker / MiRNA-210 / MiRNA-96
Positive(n=80) / Negative(n=130) / Positive(n=84) / Negative(n=126)
HYAL1 protein Positive(n=94)
Negative(n=116) / 74
6 / 20
110 / 82
2 / 12
114
2 =119.11, P :0.001** / 2 =158.19, P :0.001**
Urine marker / MiRNA-210 / ------
Positive
(n=80) / Negative
(n=130) / ------/ ------
MiRNA-96
Positive(n=84)
Negative(n=126) / 70
10 / 14
116 / ------/ ------
2 =121.49, P :0.001** / ------

Urine marker / LncRNA-UCA1
Positive(n=90) / Negative(n=120)
MiR-210
Positive(n=80)
Negative(n=130) / 74
16 / 6
114
2 =130.45, P :0.001**
Urine marker / LncRNA-UCA1
Positive(n=90) / Negative(n=120)
MiR-96
Positive(n=84)
Negative(n=126) / 70
20 / 14
106
2 =93.675, P :0.001**
Urine marker / LncRNA-UCA1
HYAL1 protein / Positive(n=90) / Negative(n=120)
Positive(n=94)
Negative(n=116) / 80
10 / 14
106
2 =124.03, P <0.001**

Supplementary Table 5s: Concordance between lncRNA-UCA1 other investigated urine markers among all groups of study (n = 210)

**Highly Significant difference was detected between investigated groups at P<0.001 using Chi-square test. miR: microRNA, lncRNA-UCA1: long non-coding RNA-Urothelial Cancer Associated 1, HYAL1: hyaluronoglucosaminidase 1.

Supplementary table 6s:Urinary Biomarkers expression among malignant and benign groups in relation to bilharziasis by chi square analysis.

HYAL1 protein / LncRNA-UCA1b / MiR-96 a / MiR-210a / Bilharsiasis / Group
Negative / Positive
(n=84) / Negative
(n=8) / Positive
(n=86) / Negative
(N=22) / Positive
(n=72) / Negative
(n=22) / Positive
(n=72) / Malignant
6(15.8%) / 32
(84.2%) / 0
(0%) / 38
(100%) / 12
(31.6%) / 26
(68.4%) / 8
(21.1%) / 30
(78.9%) / Positive
(n=38)
4(7.1%) / 52
(92.9%) / 8
(14.3%) / 48
(85.7%) / 10
(17.9%) / 46
(82.1%) / 14
(25%) / 42
(75%) / Negative
(n=56)
Negative / Positive
(n=6) / Negative
(n=56) / Positive
(n=4) / Negative
(n=52) / Positive
(n=8) / Negative
(n=52) / Positive
(n=8) / Benign
(n=54)2) / 0
(0%) / 20
(100%) / 0
(0%) / 20
(100%) / 0
(0%) / 20
(100%) / 0
(0%) / Positive (n=20)
20(100%) / 6
(15%) / 36
(90%) / 4
(10%) / 32(
80%) / 8
(20%) / 32
(80%) / 8
(20%) / Negative(n=40)

NOTE: Nonbilharzial (56; control, 40 benign, and 56malignant cases), bilharzial [20benign (bilharzial dysplasia and bilharzial cystitis) and 38malignant cases].

aStatistical difference between bilharzial and nonbilharzial benign group (2 = 4.6, P =0.043, 2 = 4.615,p=0.032 respectively).

bStatistical difference between bilharzial andnonbilharzial malignant groups at((2 = 5.9, P =0.015).

cNo statistical difference between bilharzial and nonbilharzial benign groups nor malignant groups.

Supplementary Figures

Figures Legands

Figure 1 s: Print screen Shot for miRNA-210 and UCA1 gene predictedby lnCeDBDatabase of lncRNAs acting as competing endogenous RNAs(Available at

Figure 2 s: Print screen Shot for miRNA-96 and UCA1 gene predictedby microRNA.Org Target and expression database(available at )

Figure 3 s: Print screen Shot for miRNA-210 and HYAL1 gene predictedStarBase(http://starbase.sysu.edu.cn/)

Figure 4 s: Print screen Shot for miRNA-96 and HYAL1 gene predictedby microRNA.Org Target and expression database(available at )

Figure 5 s: Print screen Shot for multiple aligned 4 sequences of the four genes and phylogenic tree thatwere generated by comparing the nucleotide sequences usingAlignX (Vector NTI Advance v. 11.5.1, Life Technologies).UCA1; Homo sapiens chromosome 19, GRCh38 Primary Assembly, HYAL1: Homo sapiens hyaluronoglucosaminidase 1 (HYAL1) mRNA, has-miR-210: Homo sapiens chromosome 11, GRCh38 Primary Assembly, has-miR-96: Homo sapiens chromosome 7, GRCh38 Primary Assembly.

Figure 6s: A subset of amplification plots for manual adjustment of the threshold according to Applied Biosystem's manual. Ideally the threshold of miRNA-210,miRNA-96, incRNA-UCA1 or or RNU-6 was set in the region where the plots are all linear and where they are all as close as possible to parallel to one another

Figure 7s : Melting curve analysis: Dye fluorescence drop rapidly when DNA melt. The figure shows dissociation curve for miR-210 and miR-96 .. Each product displays a single sharp peak indicating the specificity of the primer pairs and absence of primer-dimers or non specific amplification.

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