Tumor Biology

, Volume 36, Issue 12, pp 9545–9552 | Cite as

Integrative functional genetic-epigenetic approach for selecting genes as urine biomarkers for bladder cancer diagnosis

  • Sanaa Eissa
  • Marwa Matboli
  • Nada O. E. Essawy
  • Youssef M. Kotb
Research Article


Early screening for bladder cancer (BC) holds the key to combat and control the increasing global burden of BC mortality. We presented a simple approach to characterize, analyze, and validate a panel of biomarkers in BC and their relationship to bilharziasis. We investigated voided urine and blood samples from patients with bladder cancer (n = 94), benign bladder lesions (n = 60), and age-matched normal controls (n = 56). This study was divided into the following phases. (1) We analyzed the expression of urinary Hyaluronoglucosaminidase 1 (HYAL1) protein in BC and control samples by zymography. (2) We performed bioinformatics analysis to retrieve a set of epigenetic regulators of HYAL1. (3) This set of three selected genes [long non-coding RNA-urothelial cancer associated 1(lncRNA-UCA1), microRNA-210, and microRNA-96] was then analyzed in the same urine samples used in phase I by quantitative real-time PCR. (4) A high reproducibility of gene selection results was also determined from statistical validation. The urinary expression of HYAL1 protein and its epigenetic regulators were higher in BC patients (P < .001). The receiver-operating characteristic curve analyses demonstrated that each one had good sensitivity and specificity for distinguishing BC patients from non-BC ones (HYAL1, 89.4 and 91.2 %; miR-210, 76.6 and 93 %; miR-96, 76.6 and 89.4 %; and lncRNA-UCA1, 91.5 and 96.5 %). There was a significant positive correlation between HYAL1 and the selected epigenetic biomarkers. The performance of this urine biomarker panel reached 100 % sensitivity and 89.5 % specificity for bladder cancer diagnosis.


Bladder cancer Urine cytology Long non-coding RNA-UCA1 Bilharziasis miRNA Hyaluronoglucosaminidase 1 Urinary biomarkers 



Bladder cancer


Hyaluronoglucosaminidase 1




Long non-coding RNA




Quantitative polymerase chain reaction


Reverse transcription polymerase chain reaction


Squamous cell carcinoma


Transitional cell carcinoma


Urothelial cancer associated 1



This work was supported by Ain Shams University Research Projects 2013–14. The authors are grateful to Dr. Nahla M. Awad, Ass. Prof. of Pathology at Early Cancer Detection Unit, Faculty of Medicine, Ain Shams University, for her help in the cytological examinations of all investigated urine samples. All authors have read the journal’s policy on disclosure of potential conflicts of interest. The authors have no conflict of interest. All authors have read the journal’s authorship agreement and that the manuscript has been reviewed by and approved by all named authors.

Conflicts of interest


Authors’ contributions

Eissa S has participated in the design of the study, carried out data analysis, involved in drafting the manuscript or revising and has given final approval of the version to be published. Matboli M has performed bioinformatic analysis, practical work, participated in the design of the study, and performed the statistical analysis. Essawy N participated in the study design and involved in drafting the manuscript or revising. Youssef M. Kotb has provided us with urine, blood samples, and patient data and has given final approval of the version to be published. All authors read and approved the final manuscript.

Supplementary material

13277_2015_3722_MOESM1_ESM.doc (1.1 mb)
ESM 1 (DOC 1082 kb).


  1. 1.
    Siegel R, Jiemin M, Zhaohui Z, Jemal A. Cancer statistics 2014. A Cancer Journal for Clinicians. 2014;64(1):15422–4863.CrossRefGoogle Scholar
  2. 2.
    Sanchez C, Lachaize C, Janody F, Bellon B, Roder L, et al. Grasping at molecular interactions and genetic networks in Drosophila melanogaster using FlyNets, an Internet database. Nucleic Acids Res. 1999;27:89–94.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Lancashire LJ, Lemetre C, Ball GR. An introduction to artificial neural networks in bioinformatics—application to complex microarray and mass spectrometry datasets in cancer studies. Brief Bioinform. 2009;10:315–29.CrossRefPubMedGoogle Scholar
  4. 4.
    Fraser JR, Laurent TC, Laurent UB. Hyaluronan: its nature, distribution, functions and turnover. J Intern Med. 1997;242:27–33.CrossRefPubMedGoogle Scholar
  5. 5.
    Eissa S, Shehata H, Mansour A, Esmat M, El-Ahmady O. Detection of hyaluronidase RNA and activity in urine of schistosomal and non-schistosomal bladder cancer. Med Oncol. 2012;29:3345–51.CrossRefPubMedGoogle Scholar
  6. 6.
    Miah S, Dudziec E, Drayton RM, et al. An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer. Br J Cancer. 2012;107(1):123–8. doi: 10.1038/bjc.2012.221.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Han Y, Liu Y, Nie L, et al. Inducing cell proliferation inhibition, apoptosis, and motility reduction by silencing long noncoding ribonucleic acid metastasis-associated lung adenocarcinoma transcript 1 in urothelial carcinoma of the bladder. Urology. 2013;81(209):e1–7.Google Scholar
  8. 8.
    Noon AP, Catto JW. Noncoding RNA in bladder cancer: a specific focus upon high-risk nonmuscle invasive disease. Curr Opin Urol. 2014;24(5):506–11.CrossRefPubMedGoogle Scholar
  9. 9.
    Srivastava AK, Singh PK, Rath SK, Dalela D, Goel MM, Bhatt ML. Appraisal of diagnostic ability of UCA1 as a biomarker of carcinoma of the urinary bladder. Tumour Biol. 2014;15.Google Scholar
  10. 10.
    Qin Q, Furong W, Baosheng L. Multiple functions of hypoxia-regulated miR-210 in cancer. Cancer Res J Exp Clin. 2014;33:50. doi: 10.1186/1756-9966-33-50.CrossRefGoogle Scholar
  11. 11.
    Wang Y, Luo H, Li Y, Chen T, Wu S, Yang L. hsa-miR-96 up-regulates (MAP4K1 and IRS1 and may function as a promising diagnostic marker in human bladder urothelial carcinomas. Mol Med. 2012;5(1):260–5.Google Scholar
  12. 12.
    Jamshidian H, Hashemi M, Nowroozi MR, Ayati M, Bonyadi M, NajjaranTousi V. Sensitivity and specificity of urinary hyaluronic acid and hyaluronidase in detection of bladder transitional cell carcinoma. Urol J. 2014;11(1):1232–7.PubMedGoogle Scholar
  13. 13.
    Edge SB, Byrd DR, Compton CC, et al. AJCC cancer staging manual. 7th ed. New York: Springer; 2010. p. 497–505.Google Scholar
  14. 14.
    NCCN Clinical Practice Guidelines in Oncology: Bladder Cancer V. 2.2013. Available at September 5, 2013.
  15. 15.
    Eble JN, Sauter G, Epstein JI, Sesterhenn I, editors. World Health Organization classification of tumors pathology and genetics: tumors of the urinary system and male genital organs. Lyon: IARC Press; 2004.Google Scholar
  16. 16.
    Gui M, Idris MA, Shi YE, Muhling A, Ruppel A. Reactivity of Schistosoma japonicum and S. mansoni antigen preparations in indirect haemagglutination (IHA) with sera of patients with homologous and heterogonous schistosomiasis. Ann Trop Med Parasitol. 1991;85:599–604.CrossRefPubMedGoogle Scholar
  17. 17.
    Papageorgakopoulou N, Vynios DH, Karayanni K, Maras A, Papapetropoulou M. Electrophoretic analysis of hydrolytic enzymes of Escherichia coli cells starved in seawater and drinking water: comparison of gelatinolytic, caseinolytic, phosphohydrolytic and hyaluronolytic activities. Microbiol Res. 1997;152:299–305.CrossRefPubMedGoogle Scholar
  18. 18.
    Paraskevopoulou MD, Georgakilas G, Kostoulas Net al. DIANA-microT web server v5.0: service integration into miRNA functional analysis workflows. Nucleic Acids Res. 2013 Jul;41(Web Server issue):W169-73. Available at
  19. 19.
    Thompson JD, Higgins DG, Gibson TJ. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–80. CLUSTAL W.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C (T)) method. Methods. 2001;25:402–8.CrossRefPubMedGoogle Scholar
  21. 21.
    Nossier AI, Eissa S, Ismail MF, Hamdy MA, Azzazy HM. Direct detection of hyaluronidase in urine using cationic gold nanoparticles: a potential diagnostic test for bladder cancer. Biosens Bioelectron. 2014;54:7–14. doi: 10.1016/j.bios.2013.10.024.CrossRefPubMedGoogle Scholar
  22. 22.
    Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R. MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci U S A. 2008;105(5):1608–13. doi: 10.1073/pnas.0707594105.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Zhu L, Liu J, Cheng G. Role of microRNAs in schistosomes and schistosomiasis. Front Cell Infect Microbiol. 2014;4:165. doi: 10.3389/fcimb.2014.00165. eCollection 2014.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Sanaa Eissa
    • 1
  • Marwa Matboli
    • 1
  • Nada O. E. Essawy
    • 2
  • Youssef M. Kotb
    • 3
  1. 1.Oncology Diagnostic Unit, Medical Biochemistry and Molecular biology Department, Faculty of MedicineAin Shams UniversityCairoEgypt
  2. 2.Pharmacogenetics and Stratified MedicineUniversity College LondonLondonUK
  3. 3.Urology Department, Faculty of MedicineAin Shams UniversityCairoEgypt

Personalised recommendations