The AAPS Journal

, 20:82 | Cite as

A Non-invasive Liquid Biopsy Screening of Urine-Derived Exosomes for miRNAs as Biomarkers in Endometrial Cancer Patients

  • Akhil Srivastava
  • Katherine Moxley
  • Rachel Ruskin
  • Danny Natarajan Dhanasekaran
  • Yan Daniel Zhao
  • Rajagopal RameshEmail author
Research Article Theme: Therapeutic and Diagnostic Applications of Exosomes and other Extracellular Vesicles
Part of the following topical collections:
  1. Theme: Therapeutic and Diagnostic Applications of Exosomes and other Extracellular Vesicles


Exosomes have great potential to serve as a source of diagnostic and prognostic biomarkers for endometrial cancer (EC). Urine-derived exosomes from patients with EC and patients with symptoms of EC, but without established EC, were used to evaluate a unique miRNA expression profile. Of the 84 miRNA studied, 57 were amplified in qPCR, suggesting the differential packaging of miRNA in exosomes. Further, hsa-miR-200c-3p was identified to be enriched the most. Various bioinformatics and in silico tools were used to evaluate the biological significance of hsa-miR-200c-3p in EC. We conclude that differential miRNA in exosomes can be utilized for discovery of biomarker signatures and EC diagnosis; hsa-miR-200c-3p is one such candidate. Urine-derived exosomes pave the way for the development of non-invasive biomarkers.


exosomes endometrial cancer miRNA liquid biopsy biomarkers 



The authors thank all the patients for providing the samples and the dedicated cancer center staff for assistance in sample collection. Editorial assistance from Ms. Kathy Kyler at the office of Vice President of Research, OUHSC, is appreciated. Rajagopal Ramesh is an Oklahoma TSET Research Scholar and holds the Jim and Christy Everest Endowed Chair in Cancer Developmental Therapeutics.

Author Contributions

AS, KM, and RR conducted the studies and collected data; YDZ performed statistical analysis; AS, KM, RR, DND, YDZ, and RR conceived and designed the studies; AS and RR wrote the manuscript; AS, KM, RR, DND, YDZ, and RR critically analyzed and interpreted the data; AS, KM, RR, DND, YDZ, and RR critically reviewed, provided suggestions, and edited the manuscript; and RR supervised the project.

Funding Information

The work was supported in part by funds received from the Stephenson Cancer Center Seed Grant (RR), Presbyterian Health Foundation Seed Grant (RR), Presbyterian Health Foundation Bridge Grant (RR), Chapman Foundation, and Jim and Christy Everest Endowed Chair in Cancer Developmental Therapeutics (RR) at the University of Oklahoma Health Sciences Center.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12248_2018_220_MOESM1_ESM.docx (16 kb)
ESM 1 (DOCX 16 kb)
12248_2018_220_MOESM2_ESM.docx (15 kb)
ESM 2 (DOCX 14 kb)


  1. 1.
  2. 2.
    Fleming GF. Second-line therapy for endometrial cancer: the need for better options. J Clin Oncol Off J Am Soc Clin Oncol. 2015;33(31):3535–40.CrossRefGoogle Scholar
  3. 3.
    Fader AN, Arriba LN, Frasure HE, von Gruenigen VE. Endometrial cancer and obesity: epidemiology, biomarkers, prevention and survivorship. Gynecol Oncol. 2009;114(1):121–7.CrossRefPubMedGoogle Scholar
  4. 4.
    Akers JC, Gonda D, Kim R, Carter BS, Chen CC. Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neuro Oncol. 2013;113(1):1–11.CrossRefGoogle Scholar
  5. 5.
    Keller S, Ridinger J, Rupp A-K, Janssen JW, Altevogt P. Body fluid derived exosomes as a novel template for clinical diagnostics. J Transl Med. 2011;9:86.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006;3:1–29.Google Scholar
  7. 7.
    Vojtech L, Woo S, Hughes S, Levy C, Ballweber L, Sauteraud RP, et al. Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions. Nucleic Acids Res. 2014;42(11):7290–304.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Qazi KR, Torregrosa Paredes P, Dahlberg B, Grunewald J, Eklund A, Gabrielsson S. Proinflammatory exosomes in bronchoalveolar lavage fluid of patients with sarcoidosis. Thorax. 2010;65(11):1016–24.CrossRefPubMedGoogle Scholar
  9. 9.
    Melo SA, Sugimoto H, O'Connell JT, Kato N, Villanueva A, Vidal A, et al. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell. 2014;26(5):707–21.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    van Empel VP, De Windt LJ, da Costa Martins PA. Circulating miRNAs: reflecting or affecting cardiovascular disease? Curr Hypertens Rep. 2012;14(6):498–509.CrossRefPubMedGoogle Scholar
  11. 11.
    Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9(6):654–9.CrossRefPubMedGoogle Scholar
  12. 12.
    Whiteside TL. Tumor-derived exosomes and their role in cancer progression. Adv Clin Chem. 2016;74:103–41.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Tickner JA, Urquhart AJ, Stephenson SA, Richard DJ, O'Byrne KJ. Functions and therapeutic roles of exosomes in cancer. Front Oncol. 2014;4:127.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Azmi AS, Bao B, Sarkar FH. Exosomes in cancer development, metastasis and drug resistance: a comprehensive review. Cancer Metastasis Rev. 2013;32(3–4):623–42.CrossRefPubMedGoogle Scholar
  15. 15.
    Soung YH, Ford S, Zhang V, Chung J. Exosomes in cancer diagnostics. Cancers. 2017; 9(8): doi: 10.3390.Google Scholar
  16. 16.
    Hannafon BN, Tigoso YD, Calloway CL, Zhao YD, Lum DH, Welm AL, et al. Plasma exosome microRNAs are indicative of breast cancer. Breast Cancer Res. 2016;18(1):90.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Liu C, Eng C, Shen J, Lu Y, Takata Y, Mehdizadeh A, et al. Serum exosomal miR-4772-3p is a predictor of tumor recurrence in stage II and III colon cancer. Oncotarget. 2016;7(46):76250–60.PubMedPubMedCentralGoogle Scholar
  18. 18.
    Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol. 2008;110:13–21.CrossRefPubMedGoogle Scholar
  19. 19.
    Gilabert-Estelles J, Braza-Boils A, Ramon LA, Zorio E, Medina P, Espana F, et al. Role of microRNAs in gynecological pathology. Curr Med Chem. 2012;19(15):2406–13.CrossRefPubMedGoogle Scholar
  20. 20.
    Srivastava A, Amreddy N, Babu A, Panneerselvam J, Mehta M, Muralidharan R, et al. Nanosomes carrying doxorubicin exhibit potent anticancer activity against human lung cancer cells. Sci Rep. 2016;6:38541.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A. 2004;101(36):13368–73.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Khurana R, Ranches G, Schafferer S, Lukasser M, Rudnicki M, Mayer G, et al. Identification of urinary exosomal noncoding RNAs as novel biomarkers in chronic kidney disease. RNA. 2017;23(2):142–52.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Delić D, Eisele C, Schmid R, Baum P, Wiech F, Gerl M, et al. Urinary exosomal miRNA signature in type II diabetic nephropathy patients. PLoS One. 2016;11(3):e0150154.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Principe S, Jones EE, Kim Y, Sinha A, Nyalwidhe JO, Brooks J, et al. In-depth proteomic analyses of exosomes isolated from expressed prostatic secretions in urine. Proteomics. 2013;13(0):1667–71.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Knepper MA, Pisitkun T. Exosomes in urine: who would have thought…? Kidney Int. 2007;72(9):1043–5.CrossRefPubMedGoogle Scholar
  26. 26.
    Fernández-Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, Knepper MA. (2010). Tamm-Horsfall protein and urinary exosome isolation. Kidney Int. 2010;77(8):736–42.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Li M, Zeringer E, Barta T, Schageman J, Cheng A, Vlassov AV. Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers. Philos Trans R Soc B. 2014;369(1652):20130502. Scholar
  28. 28.
    Foj L, Ferrer F, Serra M, Arévalo A, Gavagnach M, Giménez N, et al. Exosomal and non-exosomal urinary miRNAs in prostate cancer detection and prognosis. Prostate. 2017;77(6):573–83.CrossRefPubMedGoogle Scholar
  29. 29.
    Rodríguez M, Bajo-Santos C, Hessvik NP, Lorenz S, Fromm B, Berge V, et al. Identification of non-invasive miRNAs biomarkers for prostate cancer by deep sequencing analysis of urinary exosomes. Mol Cancer. 2017;16:156.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Osamu I, Hiroshi O, Horino T, Nakamura T, Hosotani M, Mizoguchi T, et al. Urinary exosome-derived microRNAs reflecting the changes of renal function and histopathology in dogs. Sci Rep. 2017;11(7):40340.Google Scholar
  31. 31.
    Lötvall J. Hill AF. Hochberg F, Buzás EI, Di Vizio D, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. Journal of Extracellular Vesicles. 2014;3:
  32. 32.
    Wang Y, Dong X, Hu B, Wang XJ, Wang Q, Wang WL. The effects of Micro-429 on inhibition of cervical cancer cells through targeting ZEB1 and CRKL. Biomed Pharmacother. 2016;80:311–21.CrossRefPubMedGoogle Scholar
  33. 33.
    Yue S, Wang L, Zhang H, Min Y, Lou Y, Sun H, et al. miR-139-5p suppresses cancer cell migration and invasion through targeting ZEB1 and ZEB2 in GBM. Tumour Biol. 2015;36(9):6741–9.CrossRefPubMedGoogle Scholar
  34. 34.
    Sinh ND, Endo K, Miyazawa K, Saitoh M. Ets1 and ESE1 reciprocally regulate expression of ZEB1/ZEB2, dependent on ERK1/2 activity, in breast cancer cells. Cancer Sci. 2017;108(5):952–60.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Wang T, Chen X, Qiao W, Kong L, Sun D, Li Z. Transcription factor E2F1 promotes EMT by regulating ZEB2 in small cell lung cancer. BMC Cancer. 2017;17:719.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Sulaiman SA, Ab Mutalib N-S, Jamal R. miR-200c regulation of metastases in ovarian cancer: potential role in epithelial and mesenchymal transition. Frontiers in. Pharmacology. 2016;7:271.Google Scholar
  37. 37.
    Kumar S, Nag A, Mandal CC. A comprehensive review on miR-200c, a promising cancer biomarker with therapeutic potential. Curr Drug Targets. 2015;16(12):1381–403.CrossRefPubMedGoogle Scholar
  38. 38.
    Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44–57.CrossRefGoogle Scholar
  39. 39.
    Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37(1):1–13.CrossRefGoogle Scholar
  40. 40.
    Dijkhuizen FPHLJ, Mol BWJ, Brölmann HAM, Heintz APM. The accuracy of endometrial sampling in the diagnosis of patients with endometrial carcinoma and hyperplasia. Cancer. 2000;89:1765–72.CrossRefPubMedGoogle Scholar
  41. 41.
    Kirschner MA, Schneider G, Ertel NH, Worton E. Obesity, androgens, estrogens, and cancer risk. Cancer Res. 1982;42(8 Suppl):3281s–5s.PubMedGoogle Scholar
  42. 42.
    Motamedinia P, Scott AN, Bate KL, Sadeghi N, Salazar G, Shapiro E, et al. Urine exosomes for non-invasive assessment of gene expression and mutations of prostate cancer. PLoS One. 2016;11(5):e0154507.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Hessels D, Schalken JA. Urinary biomarkers for prostate cancer: a review. 2013. Asian J Androl. 2013;15(3):333–9.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Alvarez ML, Khosroheidari M, Kanchi Ravi R, DiStefano JK. Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers. Kidney Int. 2012;82(9):1024–32.CrossRefPubMedGoogle Scholar
  45. 45.
    Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002;2:569–79.CrossRefPubMedGoogle Scholar
  46. 46.
    Falcone G, Felsani A, D’Agnano I. Signaling by exosomal microRNAs in cancer. J Exp Clin Cancer Res. 2015; CR;34(1):32.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Jayaraman M, Radhakrishnan R, Mathews CA, Yan M, Husain S, Moxley KM, et al. Identification of novel diagnostic and prognostic miRNA signatures in endometrial cancer. Genes Cancer. 2017;8(5–6):566–76.PubMedPubMedCentralGoogle Scholar
  48. 48.
    Jiao A, Sui M, Zhang L, Sun P, Geng D, Zhang W, et al. MicroRNA-200c inhibits the metastasis of non-small cell lung cancer cells by targeting ZEB2, an epithelial-mesenchymal transition regulator. Mol Med Rep. 2016;13(4):3349–55.CrossRefPubMedGoogle Scholar
  49. 49.
    Jurmeister S, Baumann M, Balwierz A, Keklikoglou I, Ward A, Uhlmann S, et al. MicroRNA-200c represses migration and invasion of breast cancer cells by targeting actin-regulatory proteins FHOD1 and PPM1F. Mol Cell Biol. 2012;32(3):633–51.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Pan Y, Liang H, Chen W, Zhang H, Wang N, Wang F, et al. microRNA-200b and microRNA-200c promote colorectal cancer cell proliferation via targeting the reversion-inducing cysteine-rich protein with Kazal motifs. RNA Biol. 2015;12(3):276–89.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Hur K, Toiyama Y, Takahashi M, Balaguer F, Nagasaka T, Koike J, et al. MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis. Gut. 2013;62(9):1315–26.CrossRefPubMedGoogle Scholar
  52. 52.
    Cittelly DM, Dimitrova I, Howe EN, Cochrane DR, Jean A, Spoelstra NS, et al. Restoration of miR-200c to ovarian cancer reduces tumor burden and increases sensitivity to paclitaxel. Mol Cancer Ther. 2012;11(12):2556–65.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Ma C, Huang T, Ding Y-C, Yu W, Wang Q, Meng B, et al. microRNA-200c overexpression inhibits chemoresistance, invasion and colony formation of human pancreatic cancer stem cells. Int J Clin Exp Pathol. 2015;8(6):6533–9.PubMedPubMedCentralGoogle Scholar
  54. 54.
    Kopp F, Oak PS, Wagner E, Roidl A. miR-200c sensitizes breast cancer cells to doxorubicin treatment by decreasing TrkB and Bmi1 expression. PLoS One. 2012;7(11):e50469.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  1. 1.Department of PathologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  2. 2.Stephenson Cancer CenterUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  3. 3.Department of Gynecology OncologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  4. 4.Department of Cell BiologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  5. 5.Department of Biostatistics and EpidemiologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  6. 6.Graduate Program in Biomedical SciencesUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  7. 7.Department of PathologyStanton L. Young Biomedical Research CenterOklahoma CityUSA

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